U.S. patent application number 10/265354 was filed with the patent office on 2003-11-20 for composition and pharmaceutical preparation containing same for the treatment of herpes and related viral infections.
This patent application is currently assigned to Dalhousie University. Invention is credited to Blay, Jonathan, But, Paul P.H., Foong, Wai-Chong, Lee, Song F., Lee, Spencer H.S., Ooi, Vincent E.C., Xu, Hong-Xi, Zhang, Yongwen.
Application Number | 20030215529 10/265354 |
Document ID | / |
Family ID | 29419722 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030215529 |
Kind Code |
A1 |
Lee, Song F. ; et
al. |
November 20, 2003 |
Composition and pharmaceutical preparation containing same for the
treatment of herpes and related viral infections
Abstract
In accordance with the present invention, novel compositions
useful for the treatment of the cytopathogenic effects of an
enveloped virus in mammals have been discovered by extraction and
purification from the spikes of Prunella vulgaris. In particular,
invention compositions comprise a lignin-carbohydrate complex as an
active ingredient for inhibition of viral infection in a mammal. In
accordance with an embodiment of the present invention, it has been
discovered that invention compositions are effective agents for the
prophylaxis and therapy in mammals of diseases caused by enveloped
viruses, e.g., herpes simplex virus. Methods for producing
invention compositions and uses therefor are also provided.
Inventors: |
Lee, Song F.; (Halifax,
CA) ; But, Paul P.H.; (N.T., HK) ; Lee,
Spencer H.S.; (Halifax, CA) ; Ooi, Vincent E.C.;
(N.T., HK) ; Xu, Hong-Xi; (Halifax, CA) ;
Zhang, Yongwen; (Shatin, CN) ; Foong, Wai-Chong;
(Bedford, CA) ; Blay, Jonathan; (Bedford,
CA) |
Correspondence
Address: |
FOLEY & LARDNER
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
Dalhousie University
The Chinese University of Hong Kong
|
Family ID: |
29419722 |
Appl. No.: |
10/265354 |
Filed: |
October 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10265354 |
Oct 4, 2002 |
|
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09160210 |
Sep 23, 1998 |
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Current U.S.
Class: |
424/725 ;
514/22 |
Current CPC
Class: |
A61K 36/536
20130101 |
Class at
Publication: |
424/725 ;
514/22 |
International
Class: |
A61K 035/78 |
Claims
What is claimed is:
1. A composition comprising a lignin-carbohydrate complex, wherein
said complex inhibits viral infection of mammals.
2. The composition of claim 1, wherein the ratio of lignin to
carbohydrate in said complex is about 2:1.
3. The composition of claim 1, wherein said lignin comprises
oxidized derivatives of vanillin, syringaldehyde and
p-hydroxybenzaldehyde.
4. The composition of claim 1, wherein said carbohydrate is
characterized as a water soluble polyanionic polysaccharide
comprising glucose, galactose, xylose, arabinose, rhamnose,
mannose, and galacturonic acid, wherein glucose is a major
constituent as analyzed by paper chromatography; wherein said
carbohydrate comprises 31-35% carbon, 3-4% hydrogen, 0.5-1%
nitrogen or 2-3% sulfur; wherein about 42% carbohydrate is
expressed as glucuronic acid; and where about 7.5% uronic acid is
expressed as glucuronic acid.
5. The composition of claim 1, wherein said composition has a
molecular weight of about 8.5 kDa; wherein said composition has
little or no anti-coagulant activity as measured by the prothrombin
time test, and wherein a therapeutically effective amount of said
composition has little or no in vivo toxicity to a mammalian
subject to which it is administered.
6. The composition of claim 5, wherein said composition is stable
to temperatures in the range of about 95-100.degree. C. for 4
hours; has a pH of 5.5. when dispersed in an aqueous solution at a
concentration of about 1 mg/ml; and is substantially insoluble in
methanol, ethanol, butanol, acetone and chloroform.
7. The composition of claim 6, wherein said composition is
non-proteinaceous having a retention time of 3.56 min when
subjected to reverse-phase high pressure liquid chromatography
(HPLC) on a C18 column (25 cm.times.4.6 mm ID, 5 m, Supelcosil
LC-18, Sigma) and eluted with a mixture of 5% water and 95%
acetonitrile at a flow rate of 0.3 ml/min; binds to Alcian blue and
to DEAE Sepharose at neutral pH; and has a strong UV absorption
peak at 202 nm with a shoulder at 280 nm extending to 380 nm when
dispersed in distilled water.
8. The composition of claim 1, wherein said composition has
anti-cytopathogenic effects for an enveloped virus, and wherein
said composition inhibits viral infection both before virus binding
and after virus binding and penetration.
9. The composition of claim 8, wherein said anti-cytopathogenic
effects are anti-HSV activities.
10. The composition of claim 9, wherein said anti-HSV activities
are anti-HSV-1 activities.
11. The composition of claim 9, wherein said composition has a
direct inhibition effect to HSV-1.
12. The composition of claim 9, wherein said composition blocks the
penetration of HSV-1 into Vero cells.
13. The composition of claim 9, wherein said anti-HSV activities
are anti-HSV-2 activities.
14. The composition of claim 1, wherein said composition is derived
from the Napetoideae subfamily of plants.
15. The composition of claim 1, wherein said composition is derived
from cells of the plant Prunella vulgaris.
16. The composition of claim 1, wherein said composition is
extracted and purified from the spikes of the plant Prunella
vulgaris.
17. A pharmaceutical formulation comprising a composition of claim
1 and a pharmaceutically acceptable carrier therefor.
18. A composition comprising a lignin-carbohydrate complex, wherein
a ratio of lignin to carbohydrate is about 2:1, wherein said
complex has molecular weight of about 8.5 kDa; wherein said lignin
comprises oxidized derivatives of vanillin, syringaldehyde and
p-hydroxybenzaldehyde; wherein said carbohydrate is characterized
as a water soluble polyanionic polysaccharide comprising glucose,
galactose, xylose, arabinose, rhamnose, mannose, and galacturonic
acid, wherein glucose is a major constituent as analyzed by paper
chromatography; wherein said complex is effective for treatment of
the cytopatogenic effects of an enveloped virus in a mammal; and
wherein a therapeutically effective amount of said composition has
little or no in vivo toxicity to a mammalian subject to which it is
administered.
19. A method for producing a composition according to claim 1, said
method comprising: (a) extracting spikes of Prunella vulgaris; (b)
precipitating and purifying the extract obtained from (a); (c)
fractionating purified extract of (b); and (d) analyzing purified
fraction of (c) for antiviral activities.
20. A method for the treatment of viral infection in a mammal
comprising administering to the mammal an effective amount of a
composition of claim 17.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/160,210, filed Sep. 23, 1998, now pending,
which claims priority from U.S. Provisional Application No.
60/059,775, filed Sep. 23, 1997, both incorporated by reference
herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel composition which
can be extracted and purified from the spikes of Prunella vulgaris.
Invention composition is an effective agent for treatment of the
cytopathogenic effects of an enveloped virus in mammals. In another
aspect, the present invention relates to methods for the treatment
of the cytopathogenic effects of an enveloped virus and related
indications in mammals, employing the invention composition as the
active agent. In yet another aspect, the present invention relates
to methods for the treatment of the cytopathogenic effects of an
enveloped virus in mammals exposed to immunosuppressive regimens.
In still another aspect, the present invention relates to methods
for protecting mammals from reactivated viral infection during or
following treatment with chemotherapeutic agents. In a further
aspect, the present invention relates to methods for obtaining
invention compositions having antiviral action from Prunella
vulgaris, and formulations containing said compositions. In a still
further aspect, the present invention relates to methods of
purification of invention compositions from Prunella vulgaris and
characterization thereof. In a particular aspect, the invention
relates to compositions comprising lignin-carbohydrate complexes
having anti-HSV activities.
BACKGROUND OF THE INVENTION
[0003] Viral infections caused by enveloped viruses are a
heterogenous group of disorders characterized by viral infection of
host cells. An example of such infections includes acquired herpes
virus infections caused by broad categories of herpes-related
viruses, including the alpha-, beta- and gamma-herpes viruses, and
where the onset of infection is characterized by herpes simplex
virus (HSV) infection of host cells. Primary herpes virus
infections are normally acquired in childhood, but later enter a
dormant phase (e.g., in the nerves). Reactivation of herpes virus
infections result from a variety of factors, such as ultraviolet
light, stress, and adult onset.
[0004] Extensive work is being done to identify compounds that
inhibit the pathogenicity of viruses to prevent or treat viral
infections. While some anti-viral agents are presently available,
there is a great need for additional compounds that would be more
active, and more specific in preventing or treating various viral
infections.
[0005] The incidence and severity of herpes virus infections have
increased over the last decade as a result of disease pattern, the
frequency of drug use and the emergence of drug and analog
resistant herpes infections, resulting in new interest in the
pathophysiology of HSV enveloped virus diseases and their
progression to identify more active and more specific compounds to
prevent or treat HSV infections.
[0006] Infection with HSV-1 is a problem worldwide, regardless of
race and geography. Approximately 70% of people older than 40 have
antibodies against HSV-1 (Rawis et al., Elsevier (North-Holland)
Amsterdam, 137-152, 1981). Approximately 16-35%, 40-80%, and over
90% of the United States population are seropositive for or
infected by herpes simplex virus type 1 (HSV-1), herpes simplex
virus type 2 (HSV-2), and varicella zoster virus (VZV),
respectively. HSV-1 is the cause of cold-sores, keratitis, and
encephalitis. HSV-2 is responsible for genital herpes and VZV is
the causative agent for chicken pox and shingles. Five other
members of the herpes family, specifically the Epstein-Barr virus
(infectious mononucleosis and Burkitt's lymphoma), cytomegalovirus
(congenital CMV infections), a human herpes virus 6 and 7 (fever
and rash in children), and human herpes virus 8 (Kaposi's Sarcoma),
also cause disease in humans. In serious cases, HSV may result in
encephalitis with severe neurological sequelae and death
(Kastrukoff et al., Ann. Neurol. 22: 52-59, 1987). HSV-1 infections
in immunmocompromised patients are characterized by severe, chronic
and often extensive lesions of mucous membranes (Snoeck, Inte. J.
Antimicrobiol. Agents 16: 157-159, 2000).
[0007] Currently, two classes of antiviral drugs are utilized
clinically against herpes infections. The first class is
represented by nucleoside analogs such as acyclovir and its prodrug
derivatives (e.g. valaciclovir and famciclovir) and adenine
arabinoside (ara-A). Acyclovir inhibits viral DNA synthesis
selectively in HSV-infected cells. Acyclovir is activated by the
viral thymidine kinase and is phosphorylated to
acyclovir-triphosphate which is incorporated into the DNA chain by
the viral DNA polymerase.
[0008] The second class of antiviral drugs are direct HSV DNA
polymerase inhibitors, such as phosphonoformate (Foscovir) and
phosphonoacetate. Resistance to these drugs arises from mutation in
the thymidine kinase and in the DNA polymerase.
[0009] Xia-Ku-Cao, the fruitspikes of Prunella vulgaris L., is used
in Chinese medicine for the treatment of headache with vertigo,
acute conjunctivitis, lymph node tuberculosis, goiter mastitis and
hypertension (Chang et al., World Scientific, Singapore, p. 964,
1987). Recently, the water extract of P. vulgaris was reported to
have anti-HSV activity (Zheng, Chung His I Chieh Ho Tsa Chih 10:
39-41, 1990). Xu and coauthors reported that the polysaccharide
component in P. vulgaris had potent anti-HSV activities (Xu et al.,
Antiviral Res. 44: 43-54, 1999). The antiviral activities of P.
vulgaris have also been reported (Kageyama et al., Antiviral Chem.
Chemother. 11:157-164, 2000; Tabba et al., Antiviral Res. 11:
263-274, 1989; Yamasaki et al., Biol. Pharm. Bull. 21: 829-833,
1998).
[0010] On the other hand, Tabba et al reported isolation of an
anti-HIV component as an aqueous extract of this herb. This extract
was characterized as a sulfated polysaccharide and was named
prunellin (Tabba et al., Antiviral Res. 11: 263-274, 1989).
Subsequent studies continued to identify P. vulgaris to have
anti-HIV, HIVintegrase inhibitory, and HIV-protease inhibitory
properties (Yamazaki et al., Biol. Pharm. Bull. 21: 829-833, 1998;
Yao et al., Virology 187: 56-62, 1992; Collins, Life Sci. 60:
PL345-351, 1997; Au et al., Life Sci. 68: 1687-1694, 2001; Kageyama
et al., Antiviral Chem. Chemother. 11:157-164, 2000). Preliminary
evaluation of the mechanism of inhibition of HIV-1 infection in
vitro by purified extract of P. Vulgaris suggested that HIV-1
infection was antagonized by preventing viral attachment to the CD4
receptor (Lam et al., Life Sci. 67: 2889-2896, 2000).
[0011] Tabba et al. (Antiviral Res. 11: 263-274, 1989) and Xu et
al. (Antiviral Res. 44: 43-54, 1999) report that the bioactive
polysaccharide constituents which have the characteristics of dark
brown color or obvious ultraviolet absorption were not thought to
be "pure" polysaccharides. Moreover, in all reports, detailed
chemical composition and structural chemical properties of these
bioactive polysaceharide components have not been reported so far.
The nature of the active ingredients which are responsible for
expression of antiviral activities of P. vulgaris remains
unclear.
[0012] Over the past decade, the incidence and severity of herpes
infections have increased due to the increase in the number of
immunocompromised patients produced by aggressive chemotherapy
regimens, expanded organ transplantation, and the rising incidence
of human immunodeficiency virus (HIV) infection. This change in
disease pattern and the increase in frequency of drug use have
resulted in the emergence of acyclovir- and other nucleoside
analog-resistant herpes infections, and a need for new and useful
antiviral agents, especially those with a different mode of action
than acyclovir.
[0013] The inhibitory effects of polyanionic substances on the
replication of herpes simplex virus (HSV) and other viruses were
reported almost four decades ago. However, these observations did
not generate much interest, because the antiviral action of the
compounds was considered to be largely nonspecific. Shortly after
the identification of human immunodeficiency virus (HIV) as the
causative agent of the acquired immune deficiency syndrome (AIDS)
in 1984, heparin and other anionic (sulfated) polysaccharides were
found to be potent inhibitors of HIV-I replication in cell culture.
Since 1988, the activity spectrum of the anionic polysaccharides
has been shown to extend to various enveloped viruses, including
viruses that emerge as opportunistic pathogens (e.g., herpes
simplex virus (HSV) and cytomegalovirus (CMV)) in immunosuppressed
(e.g., AIDS) patients. As potential anti-viral drug candidates,
anionic polysaccharides offer a number of promising features.
[0014] Accordingly, there is still a need in the art for effective
compounds and methods for the prevention and treatment of the
cytopathogenic effects caused by each of the various forms of viral
infections caused by enveloped viruses.
SUMMARY OF THE INVENTION
[0015] In accordance with the present invention, pharmaceutical
compositions which can be extracted and purified from the spikes of
Prunella vulgaris are identified as effective agents for treatment
of the cytopathogenic effects of an enveloped virus in mammals.
Invention compositions have a molecular weight of less than 10 kDa,
e.g., about 8.5 kDa and comprise a lignin-carbohydrate complex
(Prunella vulgaris polysaccharide (PVP) complex) as an active
ingredient having antiviral activities. In one embodiment of the
invention, the ratio of lignin to carbohydrate in the complex is
about 2:1. The lignin in the complex is composed of vanillin,
syringaldehyde, p-hydroxybenzaldehyde and oxidation derivatives
thereof. The carbohydrate in the complex is composed of glucose,
arabinose, xylose, rhamnose, mannose, galactose, and galacturonic
acid.
[0016] In accordance with yet another embodiment of the invention,
it has been discovered that the purified compositions of the
invention are effective agents for treatment of the cytopathogenic
effects of an enveloped virus in mammals exposed to
immunosuppressive regimens. Antiviral assay and analysis of the
constituents from P. vulgaris reveal that the lignin-carbohydrate
complex of the invention composition plays an important role in the
direct inhibition of HSV-1 with an IC.sub.50 of about 18 .mu.g/ml.
Invention composition has a direct virucidal effect and impedes the
adsorption of the virus on Vero cells. It also inhibits the binding
or adsorption and penetration of virus into Vero cells and
therefore blocks cell to cell viral infection.
[0017] In accordance with yet another embodiment of the invention,
the anti-HSV activities of the invention composition on wild type
HSV-1 and the gC-deficient mutant (HSV-1 lacking the envelop
glycoprotein gC) are provided. The IC.sub.50 of the invention
composition is much lower as compared to the IC.sub.50 of heparin,
against the wild type HSV-1 and gC-deficient mutant. Thus the
invention composition is a potent anti-HSV-1 agent, as compared to
heparin. The invention composition also prevents penetration of
wild-type HSV-1 into Vero cells better than heparin. In the
gC-deficient mutant, the prevention of penetration of HSV-1 into
Vero cells by the invention composition is also observed. However,
heparin shows no effects on the prevention of penetration of
gC-deficient mutant into Vero cells. These data indicate that the
invention compositions act on the penetration effects of viral gD
protein as well as gC protein.
[0018] In accordance with yet another embodiment of the invention,
invention compositions are found to have significant in vivo
anti-HSV-1 and anti-HSV-2 activity. It is also demonstrated that 5%
and higher concentration of the invention composition in cream
formula has in vivo anti-HSV-1 and anti-HSV-2 therapeutic effects.
All these observations suggest that invention compositions are
useful as an effective anti-HSV drug.
[0019] In accordance with a further embodiment of the invention,
methods for producing invention compositions comprising
lignin-carbohydrate complex and uses thereof are provided. It has
been discovered that invention compositions can be used for
treatment of the cytopathogenic effects of an enveloped virus and
related indications in mammals by employing an effective amount of
the invention compositions as the active agent. It has also been
discovered that the purified compositions of the invention are
effective agents for protecting mammals from reactivated viral
infection during or following treatment with chemotherapeutic
agents.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 presents a representative method for extraction and
isolation of an active anti-herpes extract from the spikes of
Prunella vulgaris.
[0021] FIG. 2 presents a representative separation of active
anti-herpes materials by a Sephadex G-50 column.
[0022] FIG. 3 collectively presents the HPLC analysis of
anti-herpes preparations from Prunella vulgaris. Thus, FIG. 3A
presents the HPLC elution profile of an aqueous extract; FIG. 3B
presents the HPLC elution profile of a partially purified extract
(PVP; see Example 1), and FIG. 3C presents the HPLC elution profile
of Fraction E (see Example 3).
[0023] FIG. 4 illustrates the lack of cytotoxicity of the Prunella
vulgaris aqueous extracts up to 500 .mu.g/ml.
[0024] FIG. 5 illustrates the further separation of active
anti-herpes materials by a BioGel P4 column.
[0025] FIG. 6 presents the HPLC profile of the purified Prunella
vulgaris polysaccharide.
[0026] FIG. 7 presents a graph presenting an estimation of the
molecular mass of the Prunella vulgaris polysaccharide by HPLC.
[0027] FIG. 8 presents a gel exclusion chromatographic pattern on
Sepharose CL-6B of PVP-2, .diamond-solid., carbohydrate (490 nm);
.smallcircle., uronic acid (520 nm); .circle-solid., protein (280
nm). Vo, void volume; Vi, inner volume.
[0028] FIG. 9 illustrates the affect of protease treatment and
periodate oxidation on the anti-HSV-1 activity of PVP-2b and
various derivatives thereof. .circle-solid., PVP-2b; .box-solid.,
PVP-2b-PR (protease digested product of PVP-2b); PVP-2b-SD
(periodate oxidized product of PVP-2b). The figure shows that there
was no reduction of activity for PVP-2b-PR, in comparison with the
untreated sample PVP-2b, however, PVP-2b-SD had substantially
reduced activity.
[0029] FIG. 10 illustrates the effects of PVP-2b on adsorption of
HSV-1 into Vero cells at 37.degree. C. and 4.degree. C.
.circle-solid., 37.degree. C.; .smallcircle., 4.degree. C. The
IC.sub.50 values were found to be 7.4 .mu.g/ml at 37.degree. C. and
6.0 .mu.g/ml at 4.degree. C. The virus was inhibited significantly
by the sample at relatively high concentration at 4.degree. C. and
37.degree. C. The viral inhibition was very low for the sample at
low concentration.
[0030] FIG. 11 collectively presents immunoprecipitation results
related to isolation and identification of HSV-1 proteins that bind
to P. vulgaris polysaccharide (PVP). .sup.35S-methionine labled
HSV-1 infected (FIG. 11A) or mock-infected Vero cells (FIG. 11B)
were lysed by Nonidet P40 and sodium deoxycholate and the lysate
was applied to a PVP-Sepharose column. Following washing, the bound
proteins were eluted with 0.5% PVP. Aliquots were
immunoprecipitated by rabbit anti-gC and anti-gD antibodies.
Immuno-precipitates were recovered by Protein A-agarose beads and
subsequently analyzed by SDS-PAGE and autoradiography.
DETAILED DESCRIPTION OF THE INVENTION
[0031] In accordance with the present invention, there are provided
compositions in substantially purified form, said compositions
comprising a lignin-carbohydrate complex (PVP complex). The ratio
of lignin to carbohydrate in invention compositions is about 2:1.
The lignin in the complex is composed of oxidized derivatives of
vanillin, syringaldehyde, and p-hydroxybenzaldehyde. The
carbohydrate in the complex is characterized as:
[0032] (1) water soluble polyanionic polysaccharide(s) comprising
glucose, galactose and xylose, wherein glucose is the major
constituent as analyzed by paper chromatography, wherein the molar
ratio of glucose, relative to galactose, is at least 3 fold, and
wherein the relative abundance of the polysaccharides is
glucose>galactose.about.mannose.ab- out.galacturonic
acid>xylose.about.rhamnose.about.arabinose;
[0033] (2) having an elemental content of 30-35% carbon, preferable
31%, more prefeable 30.78%; 3-4% hydrogen, preferable 3.1%, more
preferable 3.05%; 0.5-1% nitrogen, prefeable 0.7%, more preferable
0.66%; and 2-3% sulfur, preferable 2.7%, more preferable 2.69%;
[0034] (3) containing about 42% (w/w) carbohydrate, expressed as
glucuronic acid; and
[0035] (4) containing about 7.5% (w/w) uronic acid, expressed as
glucuronic acid.
[0036] Invention compositions comprising lignin-carbohydrate
complex (PVP complex) can be further characterized as:
[0037] (1) being effective for the treatment of the cytopathogenic
effects of an enveloped virus in a mammal, and being able to
inhibit viral infection both before virus binding and after virus
binding and penetration;
[0038] (2) being non-proteinaceous and having a molecular mass less
than about 10 kDa (e.g., about 8.5 kDa);
[0039] (3) having little or no anti-coagulant activity as measured
by the prothrombin time test, and having substantially little or no
in vivo toxicity;
[0040] (4) being stable to exposure to temperatures in the range of
about 95-100.degree. C. for 4 hours and being substantially
insoluble in methanol, ethanol, butanol, acetone, and
chloroform;
[0041] (5) having a pH of 5.5 when dispersed in an aqueous solution
at a concentration of about 1 mg/ml; capable of binding to Alcian
blue and to DEAE Sepharose at neutral pH, and having a strong UV
absorption peak at 202 nm with a shoulder at 280 nm extending to
380 nm when dispersed in aqueous medium; and
[0042] (6) having a retention time of 3.56 min when subjected to
reversed-phase high pressure liquid chromatography (HPLC) on a C18
column (25 cm.times.4.6 mm ID, 5.mu., Supelcosil LC-18, Sigma) and
eluted with 5% water: 95% acetonitrile at a flow rate of 0.3
ml/min.
[0043] The novel compositions of the present invention can be
prepared by a variety of established methods for both extraction
and purification. One such method is the one described in Examples
1-3 in the present specification. In another approach, for example,
invention compositions can be obtained from cells of the plant
Prunella vulgaris, by purifying the invention composition by
contacting an extract from Prunella vulgaris with an anion exchange
material which selectively binds negatively charged materials, and
recovering the invention composition from the anion exchange
material.
[0044] The present invention is also directed to pharmaceutical
formulations suitable for the treatment of the cytopathogenic
effects of an enveloped virus in a mammal in need thereof, which
contains an effective amount of invention composition, with or
without an appropriate pharmaceutically acceptable carrier
therefor. Examples of enveloped virus include Herpes simplex virus
type 1 and 2, human immunodeficiency virus type 1 (HIV-1), human
cytomegalovirus, measles virus, mumps virus, influenza and
parainfluenza virus, respiratory syncytial virus, and the like.
[0045] The present invention is also directed to pharmaceutical
formulations suitable for the treatment of cytopathogenic effects
of an enveloped virus, more preferably herpes simplex virus, in a
mammal in need thereof, which formulation contains an effective
amount of said purified composition or an effective amount of said
purified composition together with a pharmaceutically acceptable
carrier therefor.
[0046] The present invention is also directed to methods for
treating the cytopathogenic effects of an enveloped virus which
comprises administering to a mammal in need thereof an effective
amount of the above-described pharmaceutical formulation, or of
said purified composition, optionally with a pharmaceutically
acceptable carrier. More particularly, the mammal to be treated is
a human who has been diagnosed as having an infection caused by
said enveloped virus. Most particularly the mammal to be treated is
a human who has been specifically diagnosed as having herpes
simplex virus.
[0047] In accordance with another embodiment of the present
invention, there are provided methods for the treatment of the
cytopathogenic effects of an enveloped virus in a mammal. Invention
methods comprise administering to a mammal in need thereof an
effective amount of the above-described purified composition.
[0048] As used herein, "PVP" refers to prunella vulgaris
polysaccharide. "PVP complex" refers to lignin-carbohydrate complex
according to the invention. The term "PVP complex" is
interchangeable with the terms "lignin-carbohydrate complex",
"PVP", "PVP-2b" and "PVP compound", meaning prunella vulgaris
polysaccharide complex comprising lignin and carbohydrate.
[0049] As used herein, "mammal" refers to humans as well as other
mammals, and includes animals of economic importance such as
bovine, ovine, and porcine animals. The preferred mammal
contemplated for treatment according to the invention is a human.
Adults as well as non-adults (i.e., neo-nates, pre-pubescent
mammals, and the like) are contemplated for treatment in accordance
with the invention.
[0050] As used herein, the phrase "cytopathogenic effect of an
enveloped virus" refers to the abnormal condition of a cell caused
by the infection by an enveloped virus. As noted above, viral
infections caused by enveloped viruses are a heterogenous group of
disorders characterized by viral infection in host cells. Viral
infections caused by enveloped viruses can be acquired as a direct
result of exposure to an enveloped virus, or reactivation of an
enveloped virus infection in a seropositive mammal, and which
reactivation may be induced by exposure to a broad category of
agents, including but not limited to a variety of environmental
factors (e.g., stress), immunocompromising regimens (i.e.,
pertaining to an immune response that has been weakened by a
disease or a chemotherapy agent or an immunosuppressive agent),
weakened immunocompetence arising from other viral infections (such
as HIV and the like), age onset, and the like. As described herein,
disorders characterized by viral infections in cells include
infections of cultured cells (i.e., lytic infections, persistent
infections, latent infections, transforming infections, abortive
infections, and the like), infectious disorders, conditions and
diseases (i.e., acute infections, inapparent or silent infections,
chronic and persistent infections, latent infections, slowly
progressive diseases, virus-induced tumours, and the like), and the
like.
[0051] The compositions of the present invention are preferably
present in a purified form when administered to a patient. When
invention compositions are obtained by extraction from plant
spikes, it is desirable to separate soluble extract from (residual)
particulate matter by appropriate means (e.g., filtration,
centrifugation, or other suitable separation techniques). The
utility of invention compositions as a therapeutic agent is
enhanced by greater purification. Greater dosages may be necessary
when less pure forms of the extract are employed.
[0052] Invention compositions are preferably substantially free
from heavy metals, contaminating plant materials, contaminating
microorganisms, oxalic acid or precursors of oxalic acid or any
other contaminants which may be present in a preparation which can
be derived from plant material.
[0053] Invention compositions can also be used to inhibit the
cytopathogenic effects of enveloped viruses (e.g., HSV) in mammals
in need thereof, more particularly in humans.
[0054] Invention compositions are produced by (a) extracting spikes
of Prunella vulgaris; (b) precipitating and purifying the extract
obtained from (a); (c) fractionating purified extract of (b); and
(d) analyzing purified fraction of (c) for antiviral
activities.
[0055] Although isolation of invention compositions from the spikes
of Prunella vulgaris plants is the presently most practical method
for obtaining such materials, the present invention also
contemplates obtaining such materials from other sources such as
other plants which may contain recoverable amounts of compositions
having the properties described herein. Other plants contemplated
include species within the subfamily Nepetoideae, of which Prunella
is a member. It is also possible that invention compositions could
be obtained by culturing plant cells, such as Prunella vulgaris
cells, in vitro and extracting the active ingredients from the
cells or recovering the active ingredients from the cell culture
medium.
[0056] As used herein, the term "extract" means the active
ingredients isolated from spikes or other parts of Prunella
vulgaris or other natural sources including but not limited to all
varieties, species, hybrids or genera of the plant regardless of
the exact structure of the active ingredients, form or method of
preparation or method of isolation. The term "extract" is also
intended to encompass salts, complexes and/or derivatives of the
extract which possess the above-described biological
characteristics or therapeutic indication. The term "extract" is
also intended to cover synthetically or biologically produced
analogs, homologs and mimics with the same or similar
characteristics yielding the same or similar biological effects of
the present invention.
[0057] The purified compositions contemplated for use herein
include purified extract fractions having the properties described
herein from any plant or species, preferably Prunella vulgaris, in
natural or in variant form, and from any source, whether natural,
synthetic, or recombinant. Also included within the scope of the
present invention are analogs, homologs and mimics of the
above-described purified compositions.
[0058] The present invention also contemplates the use of synthetic
preparations having the characteristics of invention compositions.
Such synthetic preparations could be prepared based on the chemical
structure and/or functional properties of the above-described
compositions of the present invention. Also contemplated are
analogs, homologs and mimics of the chemical structure of the
invention compositions and having the functional properties of
compositions according to the present invention.
[0059] As used herein, reference to "analogs, homologs and mimics"
of the invention compositions embraces compounds which differ from
the structure of invention compositions by as little as the
addition and/or replacement and/or deletion of one or more residues
thereof, to compounds which have no apparent structural similarity.
Such compounds in all instances, however, have substantially the
same activity as invention compositions. Thus, "analogs" refers to
compounds having the same basic structure as invention
compositions, but differing in several residues; "homologs" refers
to compounds which differ from invention compositions by the
addition and/or deletion and/or replacement of a limited number of
residues; and "mimics" refers to compounds which have no specific
structural similarity with respect to invention compositions.
Indeed, a mimic need not even be a polyanionic carbohydrate, but
such compound will display the biological activity characteristics
of the invention composition.
[0060] As used herein, "treatment" refers to therapeutic and
prophylactic treatment. Those in need of treatment include those
already with enveloped virus infections as well as those in which
treatment of the enveloped virus infection has failed.
[0061] Invention compositions described for use herein can be
delivered in a suitable vehicle, thereby rendering such composition
amenable to oral delivery, transdermal delivery, subcutaneous
delivery (e.g., intravenous delivery, intramuscular delivery,
intraarterial delivery, intraperitoneal delivery, and the like),
topical delivery, inhalation delivery, osmotic pump, and the like.
Depending on the mode of delivery employed, the above-described
composition can be delivered in a variety of pharmaceutically
acceptable forms. For example, the above-described composition can
be delivered in the form of a solid, solution, emulsion,
dispersion, micelle, liposome, and the like.
[0062] Pharmaceutical formulations contemplated for use in the
practice of the present invention contain invention composition in
admixture with an organic or inorganic carrier or excipient
suitable for enteral or parenteral applications. The active
ingredient may be Compounded, for example, with the usual
non-toxix, pharmaceutically acceptable carries for tablets,
pellets, capsules, suppositories, solutions, emulsions,
suspensions, and any other form suitable for use. The carriers
which can be used include glucose, lactose, gum acacia, gelatin,
mannitol, starch paste, magnesium trisilicate, talc, corn starch,
keratin, colloidal silica, potato starch, urea, medium chain length
triglycerides, dextrans, and other carriers suitable for use in
manufacturing preparations, in solid, semisolid, or liquid form. In
addition auxiliary, stabilizing, thickening and coloring agents and
perfumes may be used. The active compounds contemplated for use
herein are included in the pharmaceutical formulation in an amount
sufficient to produce the desired effect upon the target enveloped
viral disease.
[0063] Pharmaceutical formulations containing the active compound
contemplated herein may be in a form suitable for oral use, for
example, as tablets, troches, lozenges, aqueous or oily
suspensions, dispersible powders or granules, emulsions, hard or
soft capsules, or syrups or elixirs. Formulations intended for oral
use may be prepared according to any method known in the art for
the manufacture of pharmaceutical formulation. In addition, such
formulations may contain one or more agents selected from a
sweetening agent (such as sucrose, lactose, or saccharin),
flavoring agents (such as peppermint, oil of wintergreen or
cherry), colouring agents and preserving agents, and the like, in
order to provide pharmaceutically elegant and palatable
preparations. Tablets containing the active components in admixture
with non-toxic pharmaceutically acceptable excipients may also be
manufactured by known methods. The excipients used may be, for
example, (1) inert diluents such as calcium carbonate, lactose,
calcium phosphate, sodium phosphate, and the like; (2) granulating
and disintegrating agents such as corn starch, potato starch,
alginic acid, and the like; (3) binding agents such as gum
tragacanth, corn starch, gelatin, acacia, and the like; and (4)
lubricating agents such as magnesium stearate, stearic acid, talc,
and the like. The tablets may be uncoated or they may be coated by
known techniques to delay disintegration and absorption in the
gastrointestinal tract, thereby providing sustained action over a
longer period. For example, a time delay material such as glyceryl
monostearate or glyceryl distrearate may be employed. They may also
be coated by the techniques described in the U.S. Pat. Nos.
4,256,108; 4,160,452; and 4,265,874, to form osmotic therapeutic
tablets for controlled release.
[0064] In some cases, formulations for oral use may be in the form
of hard gelatin capsules wherein the active components are mixed
with an inert solid diluent, for example, calcium carbonate,
calcium phosphate, kaolin, or the like. They may also be in the
form of soft gelatin capsules wherein the active components are
mixed with water or an oil medium, for example; peanut oil, liquid
paraffin, or olive oil.
[0065] The pharmaceutical formulation may be in the form of a
sterile injectable suspension. This suspension may be formulated
according to known methods using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example,
as a solution in 1,3-butanediol. Sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic
mono- or diglycerides, fatty acids (including oleic acid),
naturally occurring vegetable oils like sesame oil, coconut oil,
peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like
ethyl oleate or the like. Buffers, preservatives, antioxidants, and
the like can be incorporated as required.
[0066] It may be desirable to administer in conjunction with the
invention composition other viral DNA synthesis inhibitors or DNA
polymerase inhibitors that promote synergistic therapeutic and
prophylactic effects.
[0067] The treatment regimen or pattern of administration of the
agents may be one with simultaneous administration of an agent
which counteracts the effects of invention compositions, and
invention compositions. In addition, the treatment regimen may be
phasic with an alternating pattern of administration of one agent
followed at a later time by the administration of the second agent.
Phasic administration includes multiple administrations of one
agent followed by multiple administrations of the second agent. The
sequence that the agents are administered in and the lengths of
each period of administration would be as deemed appropriate by the
practitioner.
[0068] Invention compositions can also be suitably administered
employing sustained-release systems. Suitable examples of
sustained-release compositions include semi-permeable polymer
matrices in the form of shaped articles, e.g. films, or
microcapsules. Sustained-release matrices include polylactides
(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556
(1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J.
Biomed. Mater. Res., 15: 267-277 (1981)), ethylene vinyl acetate
(Langer et al., supra) or poly-D-(-)-3 hydroxybutyric acid
(EP133,988), and the like. Sustained-release formulations
containing invention compositions also include liposomally
entrapped compositions according to the invention. Liposomes are
prepared by methods known in the art (see, for example, DE
3,218,121; U.S. Pat. Nos. 4,485,045 and 4,545,545).
[0069] For parenteral administration, in one embodiment, invention
compositions are formulated by mixing in a unit dosage injectable
form (solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier, i.e., one that is non-toxic to recipients at
the dosages and concentrations employed and is compatible with
other ingredients of the formulation. The formulation preferably
does not include oxidizing agents and other compounds that are
known to be deleterious to polypeptides.
[0070] Generally, the formulations are prepared by contacting
invention compositions uniformly and intimately with liquid
carriers or finely divided solid carriers or both. Then, if
necessary, the product is shaped into the desired form. Preferably
the carrier is a parenteral carrier, more preferably a solution
that is isotonic with the blood of the recipient. Examples include
water, saline, Ringers solution, dextrose solution, and the like.
Non-aqueous vehicles such as fixed oils and ethyl oleate are also
useful herein, as well as liposomes.
[0071] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as
poly-vinylpyrrolidone; amino acids, such as glycine, glutamic acid,
aspartic acid, or arginine; monosaccharides, disaccharides, and
other carbohydrates including cellulose or its derivatives,
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar
alcohols such as mannitol or sorbitol; counterions such as sodium;
and/or nonionic surfactants such as polysorbates, poloxmers, or
PEG. Invention compositions are typically formulated in such
vehicles according to clinically relevant/acceptable protocol. It
will be understood that use of certain of the foregoing excipients,
carriers, or stabilizers will result in the formation of salts of
invention compositions.
[0072] In addition, invention compositions are suitably formulated
in an acceptable carrier vehicle to form a pharmaceutical
formulation, preferably one that does not contain cells. In one
embodiment, the buffer used for formulation will depend on whether
the resulting formulation will be employed immediately upon mixing
or stored for later use. If employed immediately, invention
compositions can be formulated in mannitol, glycine, and phosphate
at an appropriate pH. If this mixture is to be stored, it is
preferably formulated in a buffer at an appropriate pH, in the
optional further presence of a surfactant that increases the
solubility of invention compositions at this pH. The final
preparation may be a stable liquid or a lyophilized solid.
[0073] While invention compositions can be formulated in any way
suitable for administration, presently preferred formulations
contain about 2-20 mg/mL of invention composition, about 2-50 mg/ml
of an osmolyte, about 1-15 mg/ml of a stabilizer, and a buffered
solution at about pH 5-6, more preferably pH about 5-5.5.
Preferably, the osmolyte is an inorganic salt at a concentration of
about 2-10 mg/ml or a sugar alcohol at a concentration of about
40-50 mg/ml, the stabilizer is benzyl alcohol or phenol, or both,
and the buffered solution is an acetate-buffered solution. Even
more prefered are formulations wherein the osmolyte is sodium
chloride and the acetic acid salt is sodium acetate. Still more
preferably, the amount of invention composition is about 8-12
mg/ml, the amount of sodium chloride is about 5-6 mg/ml, the amount
of benzyl alcohol is about 8-10 mg/ml, the amount of phenol is
about 2-3 mg/ml, and the amount of sodium acetate is about 50 mM so
that the pH is about 5.4. Additionally, the formulation can contain
about 1-5 mg/ml of a surfactant, preferably polysorbate or
poloxamer, in an amount of about 1-3 mg/ml. Alternatively, for the
preparation of invention formulation, invention composition is
suitably dissolved at 5 mg/ml in 10 mM citrate buffer and 126 mM
NaCl at pH 6.
[0074] Invention compositions to be used for therapeutic use must
be sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic compositions and formulations according to the
invention generally are placed into a container having a sterile
access port, for example, a vial having a stopper pierceable by a
hypodermic injection needle.
[0075] Invention compositions and formulations ordinarily will be
stored in unite or multi-dose containers, for example, sealed
ampules or vials, as an aqueous solution, or as a lyophilized
formulation for reconstitution. As an example of a lyophilized
formulation, 10 ml vials are filled with 5 ml of sterile-filtered
aqueous solution of composition according to the invention, and the
resulting mixture is lyophilized. The infusion solution is prepared
by reconstituting the lyophilized material in bacteriostatic
Water-for-Injection.
[0076] Typical daily doses of the active component, in general, lie
within the range of from about 10 .mu.g up to about 1g per kg body
weight, and, preferably within the range of from about 100 .mu.g up
to about 500 mg per kg body weight and can be administered up to
five times daily, for example. Presently preferred daily doses lie
within the range of from about 1 mg up to about 50 mg per kg body
weight. Typically, the active compound will be present in an amount
of 0.1 to 90 percent by weight of the pharmaceutical formulation,
preferably 0.5 to 50 percent by weight of the pharmaceutical
formulation, for example. Where one will operate within the above
ranges will vary depending on the route of administration and on a
variety of considerations, such as, for example, the age of the
patient, the size of the patient, other dysfunctions of the
patient, what, if any, other medications the patient may be taking,
and the like.
[0077] As a general proposition, the total pharmaceutically
effective amount of invention composition administered parenterally
per dose will be an amount sufficient to provide a therapeutic
effect without inducing a significant level of toxicity. Since
individual subjects may present a wide variation in severity of
symptoms and each active ingredient has its unique therapeutic
characteristics, it is up to the practitioner to determine a
subject's response to treatment and vary the dosages
accordingly.
[0078] Based on the in vitro data presented herein, a concentration
of 10 .mu.g to 50 mg of invention composition (see Example 5) per
gram of cream or ointment is expected to be effective for topical
application in preventing the cytopathogenicity of the herpes
simplex virus.
[0079] In accordance with another embodiment of the present
invention, there are provided methods for the treatment of the
cytopathogenic effects of an enveloped virus and related
indications in mammals in need thereof, said method comprising
administering an effective amount of invention composition to said
mammal.
[0080] Patients who present the cytopathogenic effects of an
enveloped virus contemplated for treatment in accordance with the
present invention are those testing seropositive to an enveloped
virus, preferably, those who are diagnosed with HSV, VZV or HIV
infections.
[0081] In accordance with yet another embodiment of the present
invention, there are provided methods for the treatment of the
cytopathogenic effects of an enveloped virus in mammals exposed to
immunosuppressive regimens, said method comprising administering an
effective amount of invention composition to said mammal.
[0082] Patients who present the cytopathogenic effects of an
enveloped virus and exposed to immunosuppressive regimens
contemplated for treatment in accordance with the present invention
are those whose normal immunotolerance is compromised by exposure
to immunosuppressive agents following, for example, organ
transplantation, xenotransplantation, subjects who have undergone
chemotherapy (e.g., cancer patients), and the like.
[0083] In accordance with still another embodiment of the present
invention, there are provided methods for protecting mammals
exposed to or following treatment with chemotherapeutic agents,
from the cytopathogenic effects of a reactivated enveloped virus
infection, said method comprising administering an effective amount
of invention composition to said mammal.
[0084] Patients for whom protection from the reactivated
cytopathogenic effects of an enveloped virus during or following
treatment to chemotherapeutic agents is indicated include patients
suffering from any disease which is commonly treated by the
administration of chemotherapeutic agents, e.g., post organ
transplant, nephrotic syndrome, cancer patients, and the like.
[0085] In accordance with a further embodiment of the present
invention, there are provided methods for preparing novel extract
fractions of Prunella vulgaris having antiviral action and
formulations containing said extract fractions. See, for example,
the methods described in the following Examples provided
herein.
[0086] The extract or extract fractions that are active in
preventing the cytopathogenic effects of an enveloped virus could
be used to treat the cytopathogenic effects of enveloped viral
infections in mammals, most preferably humans, in the following
way:
[0087] (i) Since the untreated aqueous extract has been
administered to mammalian cells up to concentrations as high as 0.5
mg/ml (see Example 14) with no apparent side effects, it could also
be ingested by human subjects and may reduce the potential for
viral associated disease.
[0088] (ii) The compound that is active against the enveloped virus
could also be administered intravenously to patients with viral
infection after the compound has been further purified. The
compound may be more effective when administered I.V. than orally
since it is unlikely that 100% of the active compound would be
absorbed from the gastrointestinal tract.
[0089] (iii) It is also conceivable that the purified compound
active against the enveloped virus could be administered into the
spinal fluid and may prevent the potential for enveloped viral
associated disease in patients with an enveloped viral
infection.
[0090] It is contemplated that the extract will be formulated into
a pharmaceutical composition comprising an effective amount of the
extract with or without a pharmaceutically acceptable carrier (as
previously described). All references and patents cited herein are
hereby incorporated by reference.
[0091] A number of natural products are known to have inhibitory
effects on herpes simplex virus. Musci and Pragai (see Experientia
41: 6 (1985)) showed the inhibitory effect of four flavonoids,
i.e., quercetin, quercitrin, rutin, and hesperidine, on HSV-1 and
Suid (alpha) HSV-1 (pseudorabies virus). A direct relationship
between viral inhibition and the ability of flavonoids to increase
cyclic AMP in the host cells was observed, suggesting flavonoids
exert their antiviral effects via cyclic nucleotide metabolism.
Wleklik et al. (see Acta Virol. 32: 522 (1988)) further showed that
hydroxylation at positions 3, 5, 7, 3', and 4' of flavonoids was
associated with the highest anti-herpes activity. Hayashi et al.
(see Antimicrob. Agents Chemother. 36: 1890-1893 (1992)) described
inhibition of HSV-1 and HSV-2 replication in Vero cells by a
bifavanone ginkgetin isolated from Cepalotaxus drupacea. The
IC.sub.50 (50% inhibition concentration) against HSV-1 was 0.91
.mu.g/ml. Ginkgetin suppressed viral protein synthesis and had no
effect on the binding and penetration of HSV-1 into cells. Barnard
et al. (see Chemother. 39:203-211 (1993)) described a flavonoid
polymer of 2100 Da that inhibited HSV penetration into cells.
[0092] Polysaccharides are known to affect the growth of animal
viruses (see Shannan, W. M. in G. J. Galasso, T. C. Merigan, and R.
A. Buchanon (ed.), Antiviral agents and viral diseases of man.
Raven Press, New York (1984) at p. 55-121). In particular, anionic
polysaccharides, such as heparin, dextran sulfate, carrageenans,
pentosan polysulfate, fucoidan, and sulfated xylogalactans, are
potent inhibitors of herpes virus binding to host cells (see, for
example, Gonalez et al. in Antimicrob. Agents Chemother. 31:
1388-1393 (1987); Baba et al. in Antimicrob. Agents Chemother. 32:
1742-1745 (1988); and Damonte et al. Chemother. 42: 57-60 (1996)).
The activity spectrum of the sulfated polysaccharides has been
shown to extend to various enveloped viruses (e.g., human
immunodeficiency virus type 1 (HIV-1), measles virus, mumps virus,
influenza and parainfluenza virus, respiratory syncytial virus
(HSV) and cytomegalovirus (CMV)), including viruses that emerge as
opportunistic pathogens. These polysaccharides are competitor of
receptors (heparan sulfate) to viral glycoproteins. Herold et al.
(see J. Virol. 70: 3451-3469 (1996) showed that N-sulfations and
the presence of carboxy groups on heparin are key determinants for
HSV-1 and HSV-2 interactions with host cells. However, these
polysaccharides also have anti-coagulant activity and are therefore
unsuitable as anti-herpes drugs.
[0093] Some plant proteins are known to have anti-herpes
activities. The better known ones are ribosome-inactivating
proteins (e.g. pokeweed anti-viral protein (see, for example,
Teltow et al. in Antimicrob. Agents Chermother. 23: 390 (1983)) and
lectins (e.g. concanavalin A; see, for example, Okada, Y., and J.
Kim. Virology 5: 507 (1972)). Pokeweed anti-viral protein is a 30
kDa single polypeptide that binds irreversibly to HSV and enters
host cells only after binding to the virus. Following entry into
the host cells, the protein inhibits protein synthesis.
Concanavalin A interacts with the viral envelope to inhibit
infectivity or to block exit of virus from infected cells. More
recently, the anti-HSV activity of two other plant proteins and one
human serum protein were described. MAP30 and GAP31 are a 30 kDa
and 31 kDa protein isolated from the Chinese bitter melon Momordica
charantia and a Himalayan tree Gelonium multiflorum, respectively
(see, for example, Bourinbaiar, A. S., and S. Lee-Huang, in
Biochem. Biophys. Res. Comm. 219: 923-929 (1996)). Both proteins
showed IC.sub.50 against HSV-1 and HSV-2 in the 0.1 to 0.5 .mu.M
range. The mode of action of MAP30 and GAP31 is not known. The high
density serum apolipoprotein A-I was found to inhibit HSV-induced
cell fusion at 1 .mu.M (see Srinivas, R. V., et al., Virology
176:48-57 (1990)). An 18 amino acid synthetic peptide analog to
apolipoprotein A-1 inhibited penetration of virus into cells but
did not prevent virus adsorption.
[0094] Other anti-herpes natural products include terpenoids and
tannins. Terpenoids (e.g. glycyrrhizic acid, see, for example,
Vanden Berghe, D. A., et al., in Bull. Inst. Pasteur 84: 101
(1986)) inhibit HSV replication (see Hudson, J. B. in Antiviral
compounds from plants. CRC Press, Inc. Boca Raton, Fla. (1990)).
Tannins are thought to inhibite viral adsorption (see, for example,
Fukudri, K., et al. in Antiviral Res. 11: 285-297 (1989)). Xu et
al. (see Heterocycles 38: 167-175 (1994)) showed that a
hydrolyzable tannin, geponin, and gallic aldehyde had IC.sub.50
against HSV-1 of 25 and 12.5 .mu.g/ml, respectively.
[0095] Prunella vulgaris, a perennial plant commonly found in
China, the British Isles, and Europe, has been used as an
astringent for internal and external purposes (see, for example,
Grieve, M. in A modern herbal. Dover Publications, NY. (1973)), as
a crude anticancer drug (see, for example, Lee, H., and J. Y. Lin.
in Mutation Res. 204: 229-234 (1988)), and as a herbal remedy to
lower high blood pressure (see, for example, Namba, T. in The
encyclopedia of Wakan-Yaku (traditional Sino-Japanese medicines),
Vol II, p. 120-121; Hoikusha Publishing Co. Ltd. Osaka, Japan
(1994)). In western herbal remedies, the plant (which is better
known as "self heal") is used in the form of hot water infusion
sweetened with honey to treat sores in the mouth and throat (see,
for example, Grieve, M. in A modern herbal. Dover Publications, NY.
(1973)). Zheng (see ChungHsi-I-Chieh-Ho-Tsa-Chih. 10: 39-41 (1990))
reported the use of a crude aqueous extract of Prunella vulgaris in
clinical treatment of herpetic keratitis with some success. Of the
78 patients who received eye drops containing crude extracts of
Prunella vulgaris and Pyrrosia lingua, 38 were reported to be
cured, 37 showed improvement, and 3 did not respond. A crude
aqueous extract of Prunella vulgaris contained no detectable
anti-coagulant activity (see Zeng, F.-Q. M. Sc. Thesis. National
University of Singapore (1996)). Hence, while there is some
evidence that Prunella vulgaris is an anti-herpes plant, the active
anti-herpes components are not known.
[0096] HSV-1, HSV-2, and VZV belong to the human alpha-herpes
viruses, while cytomegalovirus and Epstein-Barr virus belong to the
beta-herpes and gamma-herpes virus, respectively. HSV are large
(180-200 nm in diameter) enveloped viruses containing double
stranded DNA. The DNA core is surrounded by an icosahedral capsid
containing 162 capsomers. The capsid is in turn, enclosed by a
glycoprotein-containing envelope, composed of at least 11 known
glycoproteins (gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM), a
number of which are responsible for viral attachment to cells, and
the fusion of infected cells. Between the envelope and the capsid
is the tegument, which contains other viral proteins.
[0097] HSV infection of host cells is a multi-step event beginning
with binding of the virion to cell surface proteoglycans. Heparin
sulfate moieties on proteoglycans are the site of virus binding,
although HSV can also bind to chondroitin sulfate. Two viral
glycoproteins, gC and gB, are responsible for HSV attachment to
heparin sulfate. gC is the principal player in the binding and when
it is absent from the virion, gB mediates binding at a reduced
efficiency. A subsequent step in HSV entry into the cell involves
the interaction of gD with a second cell surface molecule, possibly
the mannose-6-phosphate receptor (see Brunetti, C. R., et al., J.
Biol. Chem 269: 17067-17074 (1994)). HSV penetration occurs by
fusion of the viral envelope with the cell membrane. This fusion is
pH-independent and requires the participation of at least four
viral glycoproteins, gB, gD, gH, and gL. Following the fusion,
viral nucleocapsid is released into the cytoplasm and is uncoated
to allow the viral DNA to enter the nucleus. Viral multiplication
occurs in the nucleus in an orderly fashion with the initial
appearance of immediate-early proteins necessary for regulation of
gene transcription, early proteins (e.g. DNA polymerase), and late
proteins (structural proteins). The progeny virions are widely
believed to be assembled in the nucleus and exit from the cell
through the endoplasmic reticulum.
[0098] In accordance with the present invention, extracts from more
than 20 plants have been screened for anti-HSV-1 activity using the
standard plaque reduction assay (see Edgar, L., et al. in Manual of
clinical microbiology-5th ed. A. Balows (chief ed.) American
Society for Microbiology, Washington D.C. (1991), p. 1184-1191).
The hot-water extract prepared from the spike of Prunella vulgaris
showed good activity and was not cytotoxic. A partially purified
extract (PVP) was prepared from the freeze-dried aqueous extract by
ethanol precipitation. The anti-herpes extract was further purified
by gel permeation column chromatography (Sephadex G-50). One
fraction (Fraction E) with anti-herpes activity was collected. HPLC
analysis using a reversed-phase (ODS-2) column showed that fraction
E contained one major peak and two very minor peaks. Plaque
reduction assay showed that PVP and Fraction E had an IC.sub.50
against HSV-1 of about 18 and 10 .mu.g/ml, respectively.
[0099] The purified extract was also active on clinical isolates
and acyclovir-resistant (thymidine kinase-deficient and DNA
polymerase-deficient) strains of HSV-1 and HSV-2. Preincubation of
HSV-1 with the purified extract abolished the infectivity of the
virus; however, pretreatment of Vero cells did not prevent HSV-1
infection, confirming that the extract prevented the early event
(binding and/or penetration) of infection. In a one-step growth
study, addition of PVP at 0, 2, 4, and 7.25 h after infection
showed a 99, 96, 94 and 90% reduction in total (extracellular and
intracellular) viral yield, respectively. These results confirm
that the compound also interfered with viral replication.
[0100] Fraction E contained about 42% (w/w) carbohydrate (expressed
as glucuronic acid) as determined by the phenol sulfuric acid assay
(see Dubois, M., et al. 1956. Anal. Biochem. 28:350-356 (1956)). It
contained about only 7.5% (w/w) uronic acid (expressed as
glucuronic acid) as determined by the uronic acid assay described
by Blumenkrantz and Asboe-Hansen (see Blumenkrantz, N., and G.
Asboe-Hansen in Anal. Biocehm. 54: 484-489 (1973). The compound is
polyanionic, as evidenced from its binding to Alcian blue
(Whiteman, P. in Biochem. J. 131: 343-350 (1973)) and to DEAE
Sepharose at neutral pH. Hexosamines and proteins were not
detected. As analyzed by paper chromatography, the purified
anti-herpes polysaccharide was identified to be composed of
glucose, galactose and xylose.
[0101] The purified extract was water soluble; but was insoluble in
methanol, ethanol, butanol, acetone, or chloroform. The aqueous
solution (1 mg/ml) of Fraction E had a pH of 5.5. Spectrophotometry
showed a strong absorption peak at 202 nm with a shoulder at 280 nm
which extended to 380 nm. The molecular mass of the purified
compound, estimated by HPLC with a gel filtration column, was 3,500
kDa. The polyanionic polysaccharide, prunellin, previously isolated
by Tabba et al. (see Antiviral Res. 11: 263-274(1989)) had a pH of
7.4 in aqueous solution, showed an adsorption peak at 370 nm which
extended to 500 nm, and has a molecular mass of 10,000 kDa. This
confirms that the anti-herpes compound of Fraction E contains
polyanionic carbohydrate, and is chemically different from
prunellin.
[0102] In accordance with the present invention, it has been
discovered that extracts of Prunella vulgaris are effective in the
treatment of anti-herpes viruses. The plant is known to contain
oleanolic acid, triterpene acids (ursolic acid), triterpenoids,
flavonoids (rutin), fenchone, tannins, and prunellin (see, for
example, Namba, T. in The encyclopedia of Wakan-Yaku (traditional
Sino-Japanese medicines), Vol II, p. 120-121; Hoikusha Publishing
Co. Ltd. Osaka, Japan (1994)). Prunellin is a 10 kDa anionic
polysaccharide and it has been shown to inhibit the replication of
human immunodeficiency virus-1 (see, for example, Tabba, H. D., in
Antiviral Res. 11: 263-274 (1989), and Yao, X.-J., et al. in
Virology 187: 56-62 (1992)). Rutin has been shown to have some
activity against HSV (see, for example, Mucsi, I., and B. M.
Pragai. in Experientia 41: 6 (1985)), however, none of the other
compounds have been demonstrated to have activity against herpes
viruses.
[0103] The laboratory rabbit eye and mouse ear infection models are
well characterized as experimental models in HSV infections. These
and other animal infection models can be used to study the utility
of the present extract fraction from Prunella vulgaris in
preclinical evaluations.
[0104] The Examples that follow describe extraction of Prunella
vulgaris plant spikes, characteristics of the extract and the
effect of the extract on inhibition of the cytopathogenic effects
of herpes simplex virus.
[0105] The results described in the Examples clearly show that the
P. vulgaris polysaccharide complex (PVP complex) is a more superior
anti-viral agent than known anti-herpes agents. In contrast with
heparin, a known anti-coagulant, PVP complex had an average
prothrombin time similar to that of water, indicating that PVP
complex has no or substantially no anti-coagulant activity. In
addition, PVP complex was found to inhibit HSV infection before
virus binding as well as after virus binding and penetration. The
one-sdtep growth study (Example 11) clearly shows that even after
7.25 h post virus infection, the PVP complex could reduce virus
yield by 90%. This contrasts with sodium heparin which has
anti-herpes activity only when it is present at the start of the
virus infection, i.e., only when the virus has not yet bound to the
cells to initiate the infection cycle.
[0106] The following Examples are illustrative only and are not
intended to limit the scope of the invention as defined by the
appended claims. It will be apparent to those skilled in the art
that various modifications and variations can be made in the
methods of the present invention without departing from the spirit
and scope of the invention. Thus, it is intended that the present
invention cover the modifications and variations of this invention
provided they come within the scope of the appended claims and
their equivalents.
EXAMPLES
Plant Materials & General Methods
[0107] The fruitspikes of P. vulgaris were purchased from Chinese
Medicinal Material Co. (China). A voucher specimen was
authenticated and deposited in the herbarium (No. 2399) at the
Institute of Chinese Medicine of the Chinese University of Hong
Kong. Total carbohydrate, uronic acid and protein contents were
determined by phenol-H.sub.2SO.sub.4 (Dubois et al., 1956),
m-hydroxybiphenyl (Blumenkrantz et al., 1973) and Bradford's
methods (Bradford, 1976) with Coomasie Brilliant Blue dye
(Bio-Rad), respectively, by using galactose (Gal), galacturonic
acid (GalA) and bovine serum albumin (BSA) as the respective
standards. Homogeneity of active substances was analyzed by HPLC on
combined columns (0.8.times.30 cm each) of Shodex sugar KS-850 and
KS-840 (Showa Denko Co, USA) in 0.2 M NaCl, and the fractions were
detected by an HP 1047A refractive index (RI) which was linked in
series connection with an HP 1040 A ultraviolet diode array (UV)
detector. The molecular weights of samples were estimated by
calibration curve of elution volumes of standard pullulans (P-400,
200, 100, 50, 20, 10, and 5). Lignin was analyzed by alkaline
nitrobenzene oxidation (Chen, 1988), and the resulting benzaldehyde
derivatives (vanillin, syringaldehyde and p-hydroxybenzaldehyde)
were identified by GLC-MS as the method described by Kiyohara et
al. (1999) on an HP-5 capillary column (0.32 mm i.d..times.30 m,
0.25 m film thickness, Hewlett-Packard). Helium (2 ml/min) was used
as a carrier gas, and injector and detector temperatures were 200
and 270.degree. C., respectively. Temperature program was:
60.degree. C. (1 min), 60->140.degree. C. (15.degree. C./min),
140->250.degree. C. (5.degree. C./min). Contents of lignin in
samples were colorimetrically determined by an improved acetyl
bromide method (Dence, Springer-Verlag, Berlin: 33-61, 1992),
Polysaccharides were hydrolyzed with 2 M TFA at 121.degree. C. for
1.5 h. Component sugars of the samples were analyzed as
trimethylsilyl methylglycoside derivatives by GLC (York et al.,
Methods Enzymol. 118: 3-40, 1986) on a HP-1 capillary column (0.25
mm i.d..times.30 m, 0.2 .mu.m film thickness, Supleco, USA); the
temperature program was: 140.degree. C. for 1 min,
140->180.degree. C. (2.degree. C./min), 180->275.degree. C.
(1.degree. C./min), and 275.degree. C. for 5.8 mm.
Example 1
Extraction and Fractionation of Invention Composition from Prunella
vulgaris: Method 1
[0108] Dried spikes of Prunella vulgaris (1.4 kg) were ground into
small pieces with a Waring blender. Distilled water (12 L) was
added and the suspension was simmered at 95-100.degree. C. for 90
min. The extract was decanted to a clean container and the plant
was extracted two more times with water under the same conditions.
The extracts were poured through a cotton cloth to remove insoluble
plant materials. The volume of the clarified extracts was reduced
to about 1 liter by a rotary evaporator. The condensed extract was
freeze-dried. A total of 85 g of dark brown dried powder was
obtained (see FIG. 1).
[0109] The anti-herpes component in the aqueous extract was
precipitated by ethanol. Briefly, 30 g of the freeze-dried aqueous
extract was dissolved in 300 ml of water, and ethanol was added to
a final concentration of 90% (vol/vol). The mixture was incubated
at 4.degree. C. for 18 h. The precipitate was recovered by
filtration through cotton and washed with 4.times.1.5 L of butanol,
followed by 3.times.1.5 L of methanol. This yielded 31 g of dark
brown powder, designated PVP complex (see FIG. 1). The supernatant
from ethanol precipitation contained no detectable anti-herpes
activity by the plaque reduction assay (see Example 3) and was
discarded.
Example 2
Extraction and Fractionation of Invention Composition: Method 2
[0110] The extraction process was based on a method described
previously by Zhang et al. (1997). Briefly, the fruitspikes of P.
vulgaris (500 g) were decocted for 1 h with 10 L of H.sub.2O and
the residue was decocted again for 1 h with 8 L of H.sub.2O. The
hot water extract (PVP-0, yield: 10.6%) was obtained from
lyophilization of the combined aqueous extract solution. PVP-0 was
re-dissolved in 1.5 L of H.sub.2O and then the polysaccharide
components in PVP-0 were precipitated by the addition of three
volumes of EtOH after water-insoluble materials had been removed by
centrifugation. The resulting precipitates were dissolved in water
and dialyzed against H.sub.2O for 4 days using dialysis membrane
(36 mm, Wako Chemicals Co., USA). After the non-dialyzable portion
was centrifuged to isolate the insoluble materials, the
supernatants were lyophilized to obtain a water-soluble crude
polysaccharide fraction (PVP-1, yield: 2.8%).
[0111] PVP-1 was fractionated by cetyltrimethylaminium bromide
(CTAB) method (Yamada et al., 1984) to afford the acidic
polysaccharide (PVP-2), weakly acidic polysaccharide (PVP-3) and
the neutral polysaccharide (PVP-4) fractions in a ratio (W/W) of
21:1.0:3.8. The subsequent isolation was performed in an anti-HSV-1
assay-guided way. PVP-2, which had the most potent anti-HSV-1
activity, was further fractionated by gel exclusion chromatography
on a column (2.2.times.90 cm) of Sepharose CL-6B (Pharmacia,
Sweden) using 0.2 M NaCl. One fraction near the void volume
(PVP-2a) and one fraction near the inner volume (PVP-2b) were
obtained (FIG. 8). All the obtained fractions were dialyzed against
water (yield ratios (W/W), PVP-2a: PVP-2b=1.5:1.0). These fractions
were consequently purified by fractionation on a column (2.2-90 cm)
of Sephadex 0-100 (Pharmacia, Sweden). The purified PVP-2a and
PVP-2b were assayed for their anti-HSV-1 activities by the plaque
reduction assay (see Examples 3 & 6 below). The purified PVP-2b
(PVP complex) showed potent antiviral activity with its IC.sub.50
of about 18 g/ml and PVP-2a showed no activity.
Example 3
Purification of Extract Fraction by Column Chromatography
[0112] The active anti-herpes component was further purified by gel
filtration column chromatography. An aqueous solution of PVP (450
mg in 10 ml from extraction method 1) was applied to a Sephadex
G-50 column (98.times.2.5 cm) and eluted with water. Fractions of 5
ml were collected. The anti-herpes activity was detected using the
plaque reduction assay. To do this, fractions were freeze-dried and
redissolved in distilled water to 1 mg/ml. Plaque reduction assay
was performed according to the standard method described by Edgar
et al. (see Manual of clinical microbiology-5th ed. A. Balows
(chief ed.) Amer. Soc. for Microbiology, Washington D.C. (1991), p.
1184-1191). Briefly, monolayers of Veto cells grown on culture
plates were infected with 100-200 pfu (plaque-forming unit) of
virus. After incubation for 1 h to allow viral adsorption, the
inoculum was aspirated and overlaid with medium (Dulbecco's
Modified Eagle's medium with 2% fetal calf serum) containing
dilutions of the Prunella vulgaris extract in 2% methylcellulose.
After 72 h of incubation at 37.degree. C., the plates were fixed
with formalin, stained with crystal violet; dh dried and the number
of plagues counted. Plates overlaid with medium without the extract
were used as controls. The percentage of inhibition of plaque
formation was calculated as:
[0113] (# of plagues in control)-(# of plagues in test).times.100/
(# of plaques in control)]
[0114] Active fractions were pooled and freeze-dried. The control
plates in which test compound was omitted had an average of 188.5
plaques/well.
[0115] The active anti-herpes activity was found in fractions
number 91 to number 131 (see Table 1, which presents the anti-HSV-1
activity of fractions from the Sephadex G-50 column). These
fractions were pooled to give pooled fractions C, D, E, and F with
the highest activity found in fraction E (see FIG. 2). The amount
of material recovered in each of the pooled fractions was
indicated.
1TABLE 1 Anti-HSV-1 activity of fractions from the Sephadex G-50
column % Plaque inhibition Pooled fractions Fraction No. 75
.mu.g/ml 50 .mu.g/ml 25 .mu.g/ml A (186 mg) 41 NT.sub.a 0 0 45 0 0
0 51 17.8 7.2 2.9 56 22.0 11.9 0 61 2.4 0 NT 65 11.5 2.4 NT 71 26.8
0 NT B (85 mg) 75 49.1 16.2 0 81 79.8 56.5 21.5 85 86.7 46.9 10.3 C
(58 mg) 91 100 49.6 7.7 95 100 100 11.9 101 100 100 45.9 D (42 mg)
105 100 100 65.5 111 100 100 92.0 115 100 100 82.5 E (5 mg) 121 100
100 84.6 125 100 100 100 F (32 mg) 131 100 54.9 19.4 PVP 100 73.5
36.3 .sup.aNT = not tested
[0116] The results of this example confirm that the purified
extract fraction isolated from Prunella vulgaris of the present
invention has anti-herpes activity.
Example 4
HPLC Analysis of the Purified Extract Fraction
[0117] Purity of the anti-herpes materials from Prunella vulgaris
was assessed by analyzing three preparations (aqueous extract, PVP,
and Fraction E) by reversed-phase high pressure liquid
chromatography (HPLC). Aliquots (25 .mu.l) of aqueous solutions (10
.mu.g/ml) were injected into a C18 column (25 cm.times.4.6 mm ID,
5.mu., Supelcosil LC-18, Sigma). Compounds were cluted with 5%
water: 95% acetonitrile at a flow rate of 0.3 ml/min. Compounds
were detected with a UV detector at 210 nm. The peak with a
retention time of 3.56 mm was concentrated during the purification
process (see FIG. 3). In Fraction E, this 3.56 mm peak was
essentially the only peak. Together with the anti-herpes test
results provided in Example 5 (which demonstrate an increase in
specific anti-HSV-1 activity during purification), the results of
this example confirm that the 3.56 mm peak is the purified extract
fraction of Prunella vulgaris, which purified extract fraction has
enhanced anti-herpes activity.
Example 5
Inhibitory Activity, IC.sub.50
[0118] Potency of the anti-herpes compound from Prunella vulgaris
was assessed by using a range of concentrations of PVP and Fraction
E was used to inhibit plaque formation by HSV-1 in Vero cells. The
percentage of plaque inhibition was dependent on the amount of PVP
and Fraction E added (see Table 2). IC.sub.50 is defined as the
concentration of extract that causes 50% inhibition in the number
of plaques formed in the assay system described. Zero inhibition is
where the number of plaques formed, during the assay, is equivalent
to or more than the number of plaques formed by control, where the
extract is not present. One hundred percent inhibition is where
there are no plaques formed during the assay. The concentration of
PVP and Fraction E required to give 50% inhibition (IC.sub.50) was
calculated to be about 18 and 10 .mu.g/ml, respectively. Table 2
presents dose dependent inhibition of plaque formation by PVP and
Fraction E (the control which contained no test compound had an
average of 64.25 plaques/well).
2TABLE 2 Dose dependent inhibition of plaque formation by PVP
complex and fraction E PVP (.mu.g/ml) % plaque inhibition Fraction
E (.mu.g/ml) % plaque inhibition 50 96.9 50 100 25 61.1 25 100 12.5
35.4 12.5 55.6 6.25 29.2 6.25 35.4 3.125 0 3.125 13.6
[0119] The results of this example confirm that the purified
extract fraction of the present invention isolated from Prunella
vulgaris has anti-herpes activity.
Example 6
Antiviral Activity of Invention Composition Prepared by Method
2
[0120] In vitro anti-HSV activity was assayed by plaque reduction
method described by Hill et al. (In: Manual of clinical
microbiology, 5.sup.th ed. American Society for Microbiology,
Washington, D.C., p. 1184-1191, 1991).
[0121] Cells & Virus: Vero cells (Green Monkey Kidney cells,
originally obtained from Professor K. McCarthy at the University of
Liverpool, England) used here were grown in Earle's minimum
essential medium (MEM-Earle's) at a pH of 7.2 supplemented with 10%
heat-inactivated inactiveted fetal calf serum (FCS) Two-three day
old confluent monolayer culture were prepared in 6 well culture
plate (Falcon, USA) at 37.degree. C. with 5% CO.sub.2 in a
humidified incubator. HSV-1 (acyclovir-sensitive strain, BW-S
strain, originally obtained from Jack Hill, Borroughs Wellcome Co)
was plaque-purified three times under an agarose-overlaid medium.
Viral stock was prepared by placing an inoculum of the
plaque-purified virus on Vero cell monolayers. Following adsorption
for 1 h at 37.degree. C. with 5% CO.sub.2, the virus inoculum was
removed and the cells were overlaid with maintenance medium (MM)
and incubated at 37.degree. C. with 5% CO.sub.2 for 72 h. The
infected culture medium was harvested and centrifuged for 15 min at
900 g. Supernatant was collected, aliquoted in 0.5 ml volumes and
stored at -70.degree. C. for use. The virus stock contained
1.1.times.10.sup.8laque-forming units (pfu)/ml.
[0122] Plaque Reduction Assay by Extraction Fractions: Confluent
Vero cell monolayers were grown in 6-well culture plates and
infected with 0.5 ml of MEM Earle's (supplemented with 2% FCS)
containing 200 pfu of virus. Viral adsorption was allowed for 1 h
at 37.degree. C. in a humidified atomsphere with 5% CO.sub.2 with
rocking mannually every 10 min. After removal of inculum,
monolayers were overlayed with 4 ml of serial dilutions of the p.
vulgaris extract in 0.8% methyleellulose in MM. Controls were
parallel prepared on plates overlaid with 0.8% methylcellulose in
MM without the dilutions of the p. vulgaris extract. Then the
plates were incubated at 37.degree. C. for 72 h followed by fixing
with 10% formaldehyde overnight, and staining with 1% crystal
violet in 20% ethanol. The number of plaques was counted after
air-drying. Results are expressed by percentage of plaque reduction
versus untreated controls:
[0123] n# of plagues in control)-ean# of plagues in
test).times.100/ (mean# of plaques in control)]
[0124] The anti-HSV-1 IC.sub.50 of the crude polysaccharide PVP-1
was found to be about 25 .mu.g/ml. CTAB precipitation led to three
subfractions, namely, as acidic (PVP-2), weakly acidic (PVP-3) and
neutral (PVP-4). PVP-2 showed the most potent anti-HSV-1 activity
with IC.sub.50 of about 22 .mu.g/ml whereas PVP-4 showed no
anti-HSV-1 activity. PVP-2 was fractionated on Sepharose CL-6B
column to obtain two subfractions (PVP-2a and PVP-2b). The
subfractions were subsequently purified on Sephadex G-100 column
and assayed for their anti-HSV-1 activities by plaque reduction
assay. The subfraction near the inner volume (PVP-2b) showed
significant anti-HSV activity with an IC.sub.50 of about 18
.mu.g/ml. The fraction near the void volume (PVP-2a), which was
demonstrated to be composed mainly of polysaccharides, did not show
any anti-HSV-1 activity. These results indicate that PVP-2b is the
active ingredients in PVP-2 to account for the antiviral activity
of P. vulgaris.
Example 7
Spectrum of Activity
[0125] A number of viruses were used in the plaque reduction assay
to assess the spectrum of activity of the extract fraction from
Prunella vulgaris. Results showed that PVP at 100 .mu.g/ml provided
complete inhibition of plaque formation in Vero cells by laboratory
and clinical strains of HSV-1 and HSV-2. PVP, at 100 .mu.g/ml, also
inhibited acyclovir-resistant strains of HSV-1 [strain DM2-1
(thymidine kinase-deficient) and strain PAAr5 (DNA
polymerase-deficient)] and HSV-2 strain Kost (thymidine kinase
altered). PVP at 100 .mu.g/ml showed no activity against
cytomegalovirus, human influenza virus types A and B, poliovirus
type 1, and vesicular stomatitis virus. For the test with
cytomegalovirus and human influenza viruses, human foreskin cells
and MDCK (dog kidney) cells, respectively, were used in place of
Vero cells.
[0126] The results of this example confirm that the extract
fraction from Prunella vulgarishas specific activity against HSV-1
and HSV-2 and further demonstrates that since it is active against
acyclovir-resistant HSV, the extract fraction from Prunella
vulgaris can be a useful drug of choice in treating the
cytopathogenic effects of infections caused by acyclovir-resistant
HSV.
Example 8
Effects of P-prunella vulgaris Anti-Herpes Compound on HSV-1
Infection
[0127] The mode of action of the anti-herpes effect of PVP complex
was studied. Vero cells were preincubated with 75 .mu.g/ml PVP
complex for 16 to 20 h at 37.degree. C. Cells were washed with
medium and infected with HSV-1. The same number of plaques (50
plaques/well) was observed as that in controls in which cells had
not been treated with PVP complex. This indicates that
preincubation of Vero cells with PVP complex has no protective
effect.
[0128] To study whether preincubation of virus with PVP complex
will abolish infectivity, 100 .mu.g of PVP complex was incubated
with 10.sup.5 pfu of HSV-1 at 37.degree. C. After 1 h incubation,
the mixture was diluted 10,000 fold with medium and used to infect
Vero cells. No plaque was observed on the monolayers after
incubation. In contrast, the control plate, in which the viruses
were pretreated with medium instead of PVP complex, contained an
average of 130 plagues/well. This finding indicates that the in
fectivity of HSV-1 was abolished by preincubation with PVP complex,
which probably exerts its effect by binding to the viral particles
and preventing them to bind to heparan sulfate on Vero cells. This
mode of action to reduce infectivity of herpes is completely
different from acyclovir and other known nucleoside analogs.
[0129] The protective effect of PVP complex was demonstrated by
adding PVP complex simultaneously with HSV-1 to Vera cells. When 75
.mu.g/ml of PVP complex was added at the same time with HSV-1 to
Vera cells, greater than 98% reduction in plaque formation was
observed. Incubation of 100 .mu.g PVP complex and 106
plaque-forming units of HSV at 4.degree. C., ambient temperature
(25.degree. C.) and 36.degree. C. for 1 h abrogated 99% of the
virus infectivity. The protective effect of PVP complex was further
demonstrated when PVP complex was added post infection.
[0130] The effect of PVP complex on HSV-1 growth in Vero cells was
further investigated in a one-step growth study. Monolayers of Vera
cells were infected, at 4.degree. C., with HSV-1 at a multiplicity
of infection (MOI) of 5, i.e. 5 virions per cell. At this
temperature, the virus would bind but would not penetrate the
cells. Hence, all cells in the monolayer were synchronized at the
same step of viral infection. The cells were washed with cold
medium and treated with 75 .mu.g/ml PVP complex at 0, 2, 4, and
7.25 h after the washing step. Total viral yield at each time point
was determined by plating out samples from the supernatant
(extracellular) and lyzed cells (intracellular). Results confirm
that when PVP complex was added at 0, 2, 4, and 7.25 h after
infection, the total viral yield was reduced by 99, 96, 94, and
90%, respectively, as compared to controls where cells were not
treated with PVP complex.
[0131] The results of this example confirm that the extract
fraction from Prunella vulgarisalso acts on herpes virus
intracellularly to reduce the yield of infectious virus and
prevents cell-to-cell transmission of the virus, and that the
extract fraction interferes intracellularly with certain
biosynthetic steps in the viral replication process.
Example 9
Chemical Nature of Anti-Herpes Compound from prunella vulgaris
[0132] The chemical nature of the anti-herpes compound was
investigated by different chemical tests. Total carbohydrate
content was estimated by the phenol sulfuric acid assay using
glucuronic acid as the standard, which results showed the
anti-herpes compound in Fraction E contained about 42% (w/w)
carbohydrates (expressed as glucuronic acid). Uronic acids were
measured with the method described by Blumenkrantz and Asboe-Hansen
(see Anal. Biocehm. 54: 484-489 (1973)) using glucuronic acid as
the standard. The anti-herpes compound contains about 7.5% (w/w)
uronic acid (expressed as glucuronic acid). Total hexosamines were
determined by the Molgan-Elson reagent (see Whiteman, P. in
Biochem. J. 131: 343-350 (1973)) using N-acetyiglucosamine (Sigma)
as the standard. Hexosamines were not detected. Protein was
measured by the Coomassie Blue dye binding method (Bio-Rad) using
bovine serum albumin as the standard. Protein has not been
detected. Elemental analysis showed the purified extract fraction
to contain 31-35% carbon, preferably 31%, more preferably 30.78%;
3-4% hydrogen, preferably 3.1%, more preferably 3.05; 0.5-1.0%
nitrogen, preferably 0.7%, more preferably 0.66%; and 2-3% sulfur,
preferably 2.7%, more preferably 2.69%.
[0133] The anti-herpes compound was found to be precipitated by the
cationic dye Alcian blue 8GX according to the assay method
described by Whiteman (see Biochem. J. 131: 343-350 (1973)). The
anti-herpes compound bound strongly to DEAE Sepharose at neutral pH
and could be eluted with 2 M NaCl. These experiments confirm the
anti-herpes extract fraction from Prunella vulgaris contains a
polyanionic carbohydrate.
[0134] The anti-herpes compound was water soluble, but was
insoluble in methanol, ethanol, butanol, acetone, or chloroform.
The compound is heat stable (95-100.degree. C., 4 h). A 1 mg/ml
aqueous solution of the anti-herpes compound gave a pH of 5.5.
Spectrophotometry showed a strong absorption peak at 202 nm and a
shoulder at 280 nm which extended to 380 nm. In contrast, the
prunellin previously isolated by Tabba et al (see Antiviral Res.
11: 263-274 (1989)) has a pH of 7.4 in aqueous solution, and an
absorption peak at 370 nm which extended to 500 nm.
[0135] The results of this example confirm that the anti-herpes
extract fraction from nella vulgaris different from prunellin.
Example 10
Active Constituents Analysis: Protease Digestion and Periodate
Oxidation
[0136] For determining the antiviral contribution of possible
protein moieties in the active constituents, a protease digestion
procedure was used to decompose the protein moieties. This
procedure was performed as described previously by Zhang et al
(Planta Med. 63: 393-399, 1997). PVP-2b (25 mg) was digested with
protease (6.3 mg, 9 units/mg, Sigma) in 50 mM Tris-HCl buffer (pH
7.9) containing 10 mM CaCl.sub.2 (40 ml) at 37.degree. C. for 96 h.
The reaction was terminated by neutralization with 0.1 M HCl, and
then the mixture was dialyzed against water and lyophilized to
obtain the protease digested product (PVP-2b-PR, yield: 86.0%).
[0137] For determining the antiviral contribution of carbohydrate
moieties in the active constituents, a controlled periodate
oxidation procedure was used to decompose the carbohydrate
moieties. The procedure was performed as described previously by
Zhang et al (Planta Med. 63: 393-399, 1997). PVP-2b (20 mg) was
oxidized with 50 mM NaIO.sub.4 in 50 mM acetate buffer (pH 4.5) (40
ml) at 4.degree. C. for 96 h in the dark. After the reaction had
been terminated with ethylene glycol, the product was reduced with
NaBH.sub.4 (40 mg) and dialyzed to obtain periodate oxidized
product (PVP-2b-SD, yield: 52.8%).
[0138] The crude polysaccharide fraction PVP-1 together with the
CTAB precipitation subfractions (PVP-2, 3 and 4) were compared in
their general chemical properties. The percentages of
carbohydrates, and uronic acids were calculated based on the
absorptions of the samples and equations of respective correlation
curves of galactose, and galacturouic acid (Table 3).
3TABLE 3 General properties and the anti-HSV-1 activities of the
crude polysaccharide fraction from P. vulgaris PVP-1 PVP-2 PVP-3
PVP-4 Neural sugar.sup.a (%) 52.5 62.1 85.2 98.7 Uronic acid.sup.a
(%) 25.7 22.8 8.2 3.7 IC.sub.50 (g/ml).sup.a 25 22 24 ND.sup.b
.sup.aAverage of two determinations. .sup.bND, not determined.
[0139] The subfractions (PVP-2a and 2b) of PVP-2 on gel exclusion
chromatography on Sepharose CL-6B were analyzed for their composing
sugars besides the general chemical properties. They were also
showed as single peaks by fractionation on Sephadex G-100. When
they were analyzed by HPLC on Shodex sugar KS-850+KS-840 column,
PVP-2a and PVP-2b were eluted as single peaks having respective
molecular weights of about 93 kDa and about 8.5 kDa (Table 4).
PVP-2b was found to consist of carbohydrate (12.2%) with arabinose,
xylose, rhamnose, mannose, galactose, glucose and galacturonic acid
in a molar ratio of 0.1:0.3:0.3:0.7:1.0:3.4:0.5. On the other hand,
PVP-2a was found to consist of carbohydrate (98.7%) with arabinose,
xylose, rhaninose, galactose, glucose and galacturonic acid in a
molar ratio of 4.0:11.1:0.1:1.0:4.0:1.1 as well as minor mannose.
When PVP-2b was analyzed for protein content by the Bradford
method, it seemed to contain large amounts of protein-like
substances from the Bradford-positive results (Table 4). However,
when PVP-2b was analyzed by elemental analysis, only about 3.8% of
nitrogen was found. The results suggest that PVP-2b contains no
protein.
4TABLE 4 Chemical properties of anti-HSV-1 fraction PVP-2 and its
subfractions from P. vulgaris PVP-2 PVP-2a PVP-2b Molecular weight
- 93,000 8,500 Neutral sugar (%) 62.1 98.7 12.2 Uronic acid (%)
22.8 26.2 6.6 Protein.sup.a (%) 17.4 - 17.4 Nitrogen.sup.b (%)
n.d..sup.c 3.8 Lignin (%) 13.6 n.d. 24.4 Vanillin + - +
Syringaldehyde + - + p-Hydroxybenzenaldehyde + - + Component sugar
(Mol. %) Ara - 4.0 0.1 Xyl - 11.1 0.3 Rha - 0.1 0.3 Man - n.d. 0.7
Gal - 1.0 1.0 Glc - 4.0 3.4 GalA - 1.1 0.5 .sup.aData obtained from
Bradford method. .sup.bData obtained from elemental analysis.
.sup.cn.d., not determined
[0140] In addition, UV spectrum of PVP-2b showed similar absorption
maximums at 245, 275 and 312 nm. Compared with the spectra of
commercially available substances, PVP-2b showed containing
polyphenolic compound, lignin. PVP-2b was then subjected to
alkaline nitrobenzene oxidation in order to analyze the presence of
lignin molecule, and the resulting products were analyzed by
(GLC-MS. The oxidation derivatives, vanillin, syringaldehyde, and
p-hydroxybenzaldehyde were detected in the oxidation products
derived from PVP-2b (Table 4), indicating that PVP-2b comprised
lignin moieties in addition to carbohydrate moieties. When
lignin-content in PVP-2b was calorimetrically measured by the
acetyl bromide method (Dence, 1992), PVP-2b was estimated to
contain about 24.4% of lignin (Table 4). Therefore, all evidence of
chemical properties of PVP-2b support the fact it is a
lignin-carbohydrate complex (PVP complex).
[0141] PVP-2b was treated with NaIO.sub.4 to decompose the
carbohydrate moieties in its molecules and the periodate oxidized
product PVP-2b-SD was obtained with a yield ratio of about 52.8%.
It was also treated with protease to decompose the possible protein
moieties and the protease digested product PVP-2b-PR was obtained
with a yield ratio of about 86.0%. The samples were assayed for
their anti-HSV-1 activities at the final concentrations of 100, 50,
25, 12.5 and 6.25 .mu.g/ml, by plaque reduction method. The
IC.sub.50 of PVP-2b-PR was found to be about 18 .mu.g/ml and
IC.sub.50 of PVP-2b-SD was found to be over 100 .mu.g/ml. Treatment
with periodate oxidation will reduce the anti-HSV-1 activity of
PVP-2b significantly and treatment with protease digestion did not
affect the activity through the antiviral assay of PVP-2b-SD and
PVP-2b-PR (FIG. 9). It was therefore assumed that a combination of
lignin moiety and the carbohydrate moiety in PVP-2b might be
responsible for the antiviral activity. Heparin (Sigma, USA) was
used in this assay at the final concentrations of 1000, 500, 250,
125 and 62.5 .mu.g/ml as positive control and its IC.sub.50 was
found to be 300 .mu.g/ml.
Example 11
Cytotoxicity
[0142] The cytotoxic effect of the aqueous extract on mammalian
cells was tested using a rat intestinal epithelial cell line (RIE
1) according to a published method (see Blay, J., and A. S. L. Poon
in Toxicon. 33: 739-746 (1995)). Other cell lines including those
originating from humans, such as T84 human intestinal epithelioid
cells and KB human oral epidermoid cells can also be tested to
ensure the lack of toxicity is not limited to one cell line or
species. The dried aqueous extract was dissolved in DMSO and
diluted in culture medium (Dulbecco's Modified Eagle's medium with
5% (v/v) heat-inactivated calf serum) before added directly to
RIE-1 cells. The cultures were incubated for 48 h. MTT
(3-{4,5-dimethylthiazol-2-yl]-2- ,5-diphenyl tetrazolium bromide)
was added to the culture wells to give a final concentration of 0.5
mg/ml. The cultures were incubated for 3 h at 37.degree. C. to
allow the conversion of MTT to formazan dye by mitochondrial
succinate dehydrogenase. The dye was measured at A.sub.492 with a
Titertek Multiscan plate reader. Plates containing the same amount
of DMSO, but without the test extract were used as controls. The
percent cytotoxicity was calculated by comparing the A.sub.492
readings from the tests relative to A.sub.492 readings from control
wells in which the extract fraction was omitted.
[0143] The results of this example confirm that the aqueous extract
fraction of Prunella vulgaris shows no cytotoxic effect up to the
highest concentration tested, i.e., 500 .mu.g/ml (see FIG. 4).
[0144] The series of experiments described in the above document
that invention compositions obtained from Prunella vulgaris are
lignin-carbohydrate complex (PVP complex), which are nontoxic up to
500 .mu.g/ml, and have specific novel activity against enveloped
viruses, and specifically, strains of HSV-1, HSV-2, including
acyclovir-resistant strains.
[0145] The active extract fraction from Prunella vulgaris can be
purified to homogeneity and its chemical and biological
characteristics determined in order to understand its chemical
nature, mode of action, and spectrum of activity, and thereby used
to improve its utility and potency as an antiviral drug, according
to the following examples.
Example 12
Isolation and Purification of Anti-Herpes Extract Fraction from
Prunella vulgaris
[0146] The anti-herpes anionic polysaccharide complex was purified
by re-chromatographing pooled materials from Fractions D and E (see
Example 3) on a BioGel P4 column (95.times.2.5 cm) using methods as
described. The yield and inhibitory activity against HSV-1 of
fractions obtained are shown in FIG. 5. The purified extract
fraction of the present invention can also be prepared by
extraction, precipitation, and gel filtration chromatography, as
disclosed herein. Alternative means for purification, for example,
density equilibrium centrifugation, ultrafiltration, and dialysis
can also be employed. More economical purification of the active
compound for industrial scale processes can be achieved by, for
example, ionic interaction chromatography (DEAE-Sepharose 4B) and
reversed-phase high pressure liquid chromatography. Since the
active compound binds strongly to DEAE Sepharose 4B at neutral pH
and can be eluted with 2 M NaCl, the ethanol-precipitated compounds
can be applied to a DEAE-Sepharose column, the active compound
eluted with a NaCl gradient, and the active fractions identified by
standard plaque reduction assays. Thereafter, the active fractions
can be pooled, dialyzed, and further purified by HPLC with a
preparative reversed-phase (C-18) column, a representative
purification process which can scaled up on industrial scale HPLCs.
When a sample from fraction IV was analyzed by HPLC, a single peak
was obtained, indicating the anti-herpes compound is pure (FIG. 6).
The conditions for the HPLC were as follows: column--TSK G3000
PW.times.1, 7.8 mm.times.30 cm, 6 .mu.m; flow rate of 0.8 ml/min;
mobil phase-water; detection--UV 210 nm; injection of 20 .mu.l and
a concentration of 0.2 mg/ml.
Example 13
Chemical Characterization of Anti-Herpes Compound from P.
vulgaris
[0147] The next objective was to determine the molecular weight or
mass of the purified compound. The most commonly employed methods
to determine molecular mass of macromolecules are gel permeation
chromatography, HPLC exclusion chromatography, osmotic pressure,
SDS gel electrophoresis, the Squire method using G-75, dialysis
through membranes with selected molecular mass cut-offs,
ultracentrifugation with sedimentation rate measurement, and the
like. The molecular mass of the purified compound was estimated by
HPLC with a gel filtration column (TSK-GEL G3000 PW.times.1, 7.8
mm.times.30 cm, 6 .mu.m). The purified compound has an estimated
molecular mass of 3,500 kDa. (FIG. 7; This is in contrast to the
prunellin previously isolated by Tabba et al.(Antiviral Res.
11(5-6): 263-273, 1989) which has a molecular mass of 10,000 kDa).
Elemental, Infrared, NMR and other spectroscopic analytical means
can also be employed to characterize the active compound.
[0148] The purified compound was hydrolyzed in 2 N trifluoroacetic
acid at 121.degree. C. for 1 h. When the hydrolysate was analyzed
by paper chromatography (solvent system:
pyridine:ethylacetate:water=4:10:3), three spots which have the
same R.sub.f values of glucose, galactose and xylose standards were
obtained. The product can also be exhaustively hydrolyzed in acid
(e.g., 2N HCl) and analyzed for constituent monosacharides by HPLC,
and gas chromatography. By comparing the intensity of the spots,
glucose was the major constituent sugar. Galactose and xylose were
minor components. These data were consistent with those reported in
Table 4.
[0149] These results indicate that the purified anti-herpes
polysaccharide in PVP complex is composed of mainly glucose with
some galactose and xylose as the constituent monosaccharides. Some
of these monosaccharides are likely to have SO.sub.4 and COOH
groups on them to confer the anionic nature of the polysaccharide.
Known assays, such as uronic acid assay (see Blumenkrantz, N. and
G. Asboe-Hansen in Anal. Biocehm. 54: 484-489 (1973)) and
Molgan-Elson assay (see Ghuysen, J. M., et al., in Methods
Enzymology 8: 685-699 (1966)), can be employed to verify the
amounts of uronic acid and hexosamines present. Other
characteristics of the isolated compounds, for example, pI, pH of
aqueous solution, and solubility, can be determined by means known
in the art.
Example 14
Biological Characterization of Anti-Herpes Compound from P.
vulgaris
[0150] 1. Activity of the Purified Polysaccahride Complex on
HSV
[0151] As disclosed herein, the semi-purified extract fraction has
two mechanisms of anti-HSV activity in vitro by acting
extracellularly and intracellularly against the virus. Since the
active compound(s) binds extracellularly, thereby reducing the
yield of infectious virus and preventing cell-to-cell transmission
of the virus, it is likely the compound interferes intracellularly
with other functional or biosynthetic steps in the viral
replication process.
[0152] Specific modes of action can be elucidated by using means
known in the art, for example, by the virion binding assay. In this
example, .sup.35S-labeled HSV-1 is purified by sucrose gradient
(20-60%, w/w) fractionation of culture fluids from 7-20 h
virus-infected Vero cells grown in methionine-deficient medium
containing 2% dialyzed fetal bovine serum and .sup.35S-methionine
(10 .mu.Ci/ml, specific activity >800 Ci/moles, New England
Nuclear). The radiolabeled virus is incubated with the purified
compound or medium at 4.degree. C. and then added to monolayers of
Vero cells at a multiplicity of infection (MOI) of 1. Following 1 h
incubation at 4.degree. C. to allow virus adsorption, cells are
washed free of unadsorbed viruses with phosphate-buffered saline
containing bovine serum albumin (0.5%). The extent of virus binding
can be compared by measuring the radioactivity in monolayers
infected with compound treated or medium-treated virus.
Alternatively, monolayer cells can be solubilized with a detergent,
and the cell-bound radiolabel analyzed by SDS-PAGE and
fluorography.
[0153] The major viral capsid protein VP5 can be used as a
convenient protein for densitometric quantitation (see Herold, B.
C., et al., J. Virol. 65: 1090-1098, 1991). Other binding
experiments known in the art can be employed to study the effect on
invention compound on virus binding kinetics, and on virus already
bound to Vero cells.
[0154] The specific role of the Prunella vulgaris compound in virus
binding in relationship to gC and gB can be further validated using
gC-deficient and gB-deficient mutants of HSV-1 and specific anti-gC
and gB antibodies. The effect of the compound on virus penetration
can be studied using known methods in the art, for example, Herold
et al., J. Virol. 22: 3461-3469, 1996), which determines the
resistancy of an adsorbed virus to a pH 3.0 buffer.
[0155] Intracellular anti-HSV activity of the compound can be
studied by infecting Vero cells with HSV-1 at a MOI of 20 at
4.degree. C., and subsequent virus growth (from extracellular
supernatants and cell lysates) in the presence or absence of the
compound then compared at 0, 0.5, 1, 2, 4, 8, 12, 16, and 20 h
post-infection at 37.degree. C. This example of a "single-cell
growth" experiment is expected to confirm the invention results
disclosed herein, specifically, that the addition of the compound
following infection reduces virus yields in cultures.
Ultrastructural studies on these samples can be performed using
known methods (see, for example, Gollins and Porterfield, J. Gen.
Virol. 66: 1969-1982 (1985). Comparison of the mode of entry of
HSV-1 into cells, with the subsequent morphological development in
the cellular and subcellular compartments of infected cells
incubated in the presence or absence of the compound, will lead to
increased knowledge of the fundamental viral inhibitory
characteristics of invention compound and can be used to define the
mode of action of invention compound at the biochemical and
molecular level.
5TABLE 5 Activity of the Purified Polysaccharide on HSV % Plaque
Inhibition Acyclovir 50 25 12.5 6.25 Virus sensitivity Strain
#.sup.a .mu.g/ml.sup.b .mu.g/ml .mu.g/ml .mu.g/ml HSV-1 Sensitive
15577 100 95 49 0 HSV-1 Resistant 12959 100 100 14 0 Thymidine
kinase deficient HSV-1 Resistant 15518 100 88 20 0 Thymide kinase
altered HSV-2 Sensitive 15614 100 100 41 18 HSV-2 Resistant 15597
100 95 49 0 Thymidine kinase altered .sup.aWell-characterized
strains from Burroughs Wellcome Co. .sup.bConcentrations of the
purified anti-herpes compound used.
[0156] These results show that the purified polysaccharide complex
from P. vulgaris contains activity against both types of herpes
viruses regardless of whether they are sensitive or resistant to
acyclovir. The results indicate that the PVP complex has a
different mode of action than acyclovir and imply that it can be
used for treatment of infections caused by acyclovir-resistant
herpes viruses.
[0157] The activity of the purified compound of the extract
fraction can be tested against viruses in the herpes family (e.g.
VZV and cytomegalovirus) and other enveloped viruses, such as human
immunodeficiency virus type 1 (HIV-1), human cytomegalovirus,
virus, and the like, to determine its full spectrum of activity and
anti-coagulant activity, useful for determining its specific
utility for a given enveloped virus.
[0158] 2. Anti-Coagulant Activity
[0159] The anti-coagulant activity of the PVP complex was measured
by the prothrombin time test. The prothrombin times were measured
at 37.degree. C. using a BBL fibrometer (Becton Dickinson and Co,
USA). Blood (9 ml), collected from the rabbit marginal ear vein,
was mixed with 3.8% sodium citrate (1 ml) and centrifuged at
1,500.times.g for 10 min at room temperature to obtain a clear
supernatant as the testing plasma. In a typical assay, the mixture
containing 50 .mu.l of the testing solution (1 mg/ml), 150 .mu.l of
50 mM Tris buffer, 0.1 M HCl, pH 7.5, 100 .mu.l of thromboplastin
with calcium and 100 .mu.L of plasma, was incubated at 37.degree.
C. The prothrombin. times were recorded in seconds as the
fibrometer stopped due to clotting.
[0160] The average prothrombin time for the anti-herpes PVP complex
was 25.9.+-.1.5 seconds, a value similar to the water control
29.9.+-.1.4 seconds. Water is employed as a control to show the
normal prothrombin time of the plasma. The results showed that
anti-herpes PVP complex has substantially little or no
anti-coagulant activity. This is in contrast to a known
anti-coagulant anionic polysaccharide, heparin, which has also been
described to have anti-HSV activity (Herold et al., J. Virol. 22:
3461-3469, 1996). The probrombin time for anti-coagulant (e.g.,
heparin) is about 300 seconds.
[0161] 3. Comparative Studies of the Anti-Herpes Activity Between
the P. vulgaris Compound and Sodium Heparin.
[0162] The anti-herpes activity of the PVP complex and sodium
heparin was studied to illustrate the differences between the two.
First, using the plaque reduction assay as described in Examples 3
& 6, a significant difference in the ability to inhibit HSV-1
strain #15577 and strain Delta gC2-3 (a glycoprotein C deficient
HSV-1, Herold et al., J. Gen. Virol. 75: 1211-1222, 1994)) was
observed. Glycoprotein C is one of the viral envelop proteins that
mediates virus binding to heparin receptors on mammalian cells.
Heparin is thought to inhibit HSV-1 infection of cells by competing
with heparin receptors (Harold et al., J. Gen. Virol. 75:
1211-1222, 1994)). The IC.sub.50 of heparin against strains 15577
and Delta gC2-3 were estimated as 750 .mu.g/ml and >1 mg/ml,
respectively. In contrast, the IC.sub.50 of the PVP complex against
strains #15577 and Delta gC2-3 were estimated as 10 .mu.g/ml and 5
.mu.g/ml, respectively.
[0163] A second major difference between the PVP complex and sodium
heparin was observed by the binding experiment. In this experiment,
0.1 ml of HSV-1 (10.sup.5 pfu) was incubated with 0.1 ml of sodium
heparin (20 mg/ml), or the PVP complex (1 mg/ml), or water at
36.degree. C. for 1 h. The mixture was serially diluted in medium
and the number of residual infectious virus was determined by the
plaque assay. The number of plaque developed in the water treatment
control was taken as 100%. The results showed that after the
treatment with 2 mg of heparin, 30% and 42% of the original HSV-1
strains #15577 and Delta gC2-3, respectively, were still infectious
and recovered by the plaque assay. In contrast, the same amount of
virus after exposure to 100 .mu.g of the PVP complex, none of the
virus from both strains remained infectious and could not be
recovered by the plaque assay.
[0164] The anti-herpes effect of sodium heparin was further
investigated by a binding competition experiment. In this
experiment, 0.5 ml samples of HSV-1 (about 100 pfu) were mixed with
0.5 ml of sodium heparin at different concentrations. The mixtures
were immediately used to infect Vero cells at 37.degree. C. for 1
h. Following the incubation, the number of plaques were determined
in the plaque assay. The results showed that there was a 88%
inhibition of plaque formation for strain #15577 when 62.5 .mu.g/ml
heparin was used. However, complete inhibition of plaque formation
could not be achieved even when 1 mg/ml heparin was used. At 1
mg/ml heparin, the % plaque inhibition was 92%. Similar results
were obtained when the gC-deficient HSV-1, Delta gC2-3, was used.
In which case, the % plaque inhibition for 62.5 .mu.g/ml and 1
mg/ml heparin were 77% and 86%, respectively.
[0165] These results suggest that sodium heparin has anti-herpes
activity only when it is present at the start of the virus
infection. In other words, heparin can have inhibitory effect on
HSV only when the virus has not yet bound to the cells to initiate
the infection cycle. This statement is further supported by the
high IC.sub.50 values (750-1000 .mu.g/ml) as determined by the
plaque reduction assay. In the plaque reduction assay, heparin was
added 1 h after the virus has incubated (i.e. has bound and
probably penetrated the cells) with the vero cells. Heparin has no
inhibitory effect as indicated by the high IC.sub.50 values. The
anionic polysaccharide heparin exerts its anti-herpes effect by
blocking virus binding to the cells. This blocking can only be
effective when the virus has not bound to the cell receptors. This
is contrast to the P. vulgaris anionic polysaccharide complex that
it can inhibit HSV infection before virus binding as well as after
virus binding and penetration. The results described in the
one-step growth study (Example 8) clearly show that even after 7.25
h post virus infection, the P. vulgaris polysaccharide complex
could reduce virus yield by 90%. In addition, the results described
in this example, specifically in the plaque reduction assay and the
binding experiment, clearly show that the P. vulgaris
polysaccharide complex is a superior anti-herpes agent relative to
heparin.
Example 15
Virucidal Effects of PVP Complex
[0166] Virucidal effect assay was adapted from the method described
previously by Xu et al. (Antiviral Res. 44: 43-54, 1999). 0.1 ml of
HSV-1 containing 4.4.times.10.sup.6 pfu was incubated with 0.1 ml
of tested sample in serum-free MEM (pH 7.4) at 37.degree. C. for 1
h. Controls were made by mixing 0.1 ml of HSV-1 with 0.1 ml of
medium. The treated virus was promptly 10.sup.5-fold diluted with
MEM supplemented with FCS and assayed for infectivity by plaque
reduction method on 6-well plate.
[0167] Virusidal effect was assayed by direct incubation of HSV-1
with tested sample. The virus was diluted after pretreatment with
the sample and assayed for infectivity by plaque reduction method.
In PVP-2b group, it is provided that the 85% of the viruses were
inhibited in comparison with the medium treated control, suggesting
that PVP-2b has the direct inhibition effect to HSV-1.
Example 16
Assays for Effect of PVP Complex on Virus Binding to Vero Cells
[0168] HSV-1 stock was diluted to 100 pfu in prechilled MM and
mixed with an equal volume of prechilled MM containing 50, 25,
12.5, 6.25 and 3.125 .mu.g/ml of the sample or with only MM for
control. The mixtures in 250 .mu.l were immediately inoculated on
Vero cell cultures at 4.degree. C. and 37.degree. C. After
adsorption for 1 h, the inoeula were removed from the culture
followed by washing twice with the medium. Plaque formation was
allowed by incubation at 37.degree. C. for 72 h in 0.6% agrose
medium.
[0169] Effect of PVP-2b on virus adsorption to Vero cells was
investigated at concentrations of 50 .mu.g/ml to 3.125 .mu.g/ml at
4.degree. C. and 37.degree. C. A dose-dependent impeding effect to
virus adsorption to Vero cells was observed at 4.degree. C.
However, a similar trend of dose-dependent impeding effect to virus
adsorption was also observed at 37.degree. C. In this assay, PVP-2b
showed the significant blocking effect to HSV-1 adsorption to Vero
cells at 4.degree. C. and 37.degree. C. after pretreating the virus
with the sample. The IC.sub.50 were found to be 7.4 .mu.g/ml at
37.degree. C. and 6.0 .mu.g/ml at 4.degree. C. Effects of PVP-2b on
virus adsorption to Vero cells were not found at 3.125 .mu.g/ml
(FIG. 10).
Example 17
Effects of PVP Complex on Penetration of HSV-1 into Vero Cells
[0170] Penetration Assay. The penetration assay was conducted using
the method as described in Example with modifications. Confluent
Vero cell monolayers in 25 ml tissue culture flasks were chilled to
4.degree. C. for 45 mm. The monolayers were infected with 300 pfu
of HSV-1 in 2.5 ml of cold MEM Earle's medium. Attachment was
synchronized for 1 h at 4.degree. C. The flasks were washed twice
with 4 ml of cold PBS to remove any unbound virus. The monolayers
were covered with 5 ml of maintenance medium and shifted to
37.degree. C. At set time intervals, the medium was removed and the
cells were treated one of the following for ways: (a) PVP complex,
(b) heparin, (c) citrate buffer, pH 3 (80 mM ciric acid, 40 mM
Na.sub.2HPO.sub.4) and no treatment control. For the PVP- or
heparin-treated groups, 5 ml of fresh maintenance medium containing
100 .mu.g/ml PVP complex or 1000 .mu.g/ml heparin was added to the
monolayers. The flasks were incubated at 37.degree. C. for 90 min,
after which the monolayers were washed twice with 4 ml of PBS and
covered with 10 ml of 0.8% methylcellulose in maintenance medium.
Plaque formation was allowed at 37.degree. C. for 72 h. For the
citrate buffer treated group, 3 ml of buffer was added to the
monolayers for 1 min. The cells were washed twice with PBS, after
which medium was added to allow plaque formation. For the no
treatment control group, following medium removal, the monolayers
were washed twice with PBS and medium added to allow plaque
formation.
[0171] Effects of PVP complex on HSV penetration into Vero cells.
Tables 6 and 7 showed the effect of PVP on wild-type HSV-1 and
gC-deficient HSV-1 mutant penetration into Vero cell. Citrate
buffer at pH 3 represents the penetration kinetics of HSV into Vero
cells. The low pH removes adherent virus particles from the cell
surface (i.e., those that have not yet penetrated). At time 20 min,
all the wild type virus particles have penetrated and can not be
removed by the low pH buffer (Table 6). When the cultures were
treated with PVP complex, a reduction in plaque formation with
respect to time of PVP complex addition was observed. This
reduction is less obvious than the heparin treatment. At time 0
min, only 19% of the virus could penetrate and form plaque as
compared to 49% in the heparin treatment.
[0172] In the case of gC-deficient mutant, the rate of penetration
was slower than that displayed by the wild-type HSV (Table 7).
However, by 60 min, all the virus particles appeared to have
penetrated. The prevention of penetration this mutant by PVP
complex was again observed. In contrast, heparin had no apparent
effects on the penetration of the gC-deficient mutant into Vero
cells. The data indicates that PVP complex can prevent penetration
of wild-type HSV-1 better than heparin. The effect is even more
clear in the case of the gC-deficient mutant and this suggests that
PVP complex acts on gD viral protein in addition to gC (FIG.
11).
6TABLE 6 Effect of PVP complex and heparin on HSV-1 penetration
into vero cells % Plaques PVP Heparin Citrate buffer, No treatment
Time (min) (100 .mu.g/ml) (1000 .mu.g/ml) (pH 3) control 0 19 49 0
96 2 38 57 12 92 4 48 88 32 100 6 53 86 50 100 10 80 100 78 91 15
95 100 82 95 20 100 100 100 100
[0173]
7TABLE 7 Effect of PVP complex and heparin on the glycoprotein
C-deficient mutant HSV-1 penetration into vero cells. % Plaques PVP
Heparin Citrate buffer No treatment Time (min) (100 .mu.g/ml) (1000
.mu.g/ml) (pH 3) control 0 6 62 0 89 10 15 90 0 91 20 17 100 0 100
30 24 100 0 100 40 48 100 18 100 50 91 84 28 100 60 100 100 100
100
Example 18
Identification of gC and gD as the PVP Complex Binding Target
[0174] Radiolabelling of HSV. Vero cell monolayers grown on tissue
culture dishes (100.times.20 mm, Falcon #35-3003) was strayed of
methionine by incubating for 1 h at 37.degree. C. in MEM-Earle's
methionine-free medium supplemented with 2% dialysed fetal calf
serum. The monolayers were then chilled at 4.degree. C. for 1 h,
after which were infected with the HSV-1 at a multiplicity of
infection of 5. The viral adsorption was allowed to occur at
4.degree. C. for 1 h. The monolayers were then washed twice with
methionine-free maintenance medium. Eight ml of the same medium
containing 275 .mu.Ci of .sup.35S-methionine (NEN, specific
activity 1175 Ci/mmol) was added to each plate. The cultures were
labeled for 24 h, at which time the cells were detached with a
rubber policeman and harvested by centrifugation (3,000.times.g, 10
min). The cell pellets were frozen at -20.degree. C. Parallel
monolayers that have not been infected with HSV were similarly
radiolabeled and used as the mock-infected control. The cell
pellets were lyzed with three freeze-thaw cycles and 1 ml of
phosphate buffered saline containing 0.5% nonidet-P40 and
deoxycholate (PBS-NP40-DOC) was added. After incubation on ice for
30 min and the lysate was used immediately in affinity
chromatography.
[0175] Affinity chromatography. PVP-Sepharose beads were prepared
by coupling PVP to CNBr-activated Sepharose (Pharmacia). The
PVP-Sepharose column (2 ml bed volume) was equilibrated with 20 ml
of PBS-NP40-DOC prior to use. HSV-1 infected or mock-infected Vero
cell lysates in 1 ml of PBS-NP40-DOC was added to the column and
incubated at room temperature for 5 min. The column was washed with
20 ml of PBS-NP40-DOC. Bound proteins were eluted with 10 ml of 5
mg/ml PVP complex in PBS-NP40-DOC. The proteins in the eluate were
precipitated with 5% trichloroacetic acid (TCA), washed three times
with cold acetone, and dissolved in 55 .mu.l of Tris-SDS buffer
(0.5 M Tris, pH 8, and 4% SDS).
[0176] Immuno-precipitation. The samples from affinity
chromatography were boiled for 5 min and centrifuged
(10,000.times.g, 5 min). A 5 .mu.l aliquot was removed as total
protein from PVP complex eluate. The remaining 50 .mu.l sample was
divided into two 25 .mu.l aliquots. One ml of Triton X-100 buffer
(50 mM Tris, pH 7.7, 0.15 M NaCl, 5 mM EDTA, and 1% Triton X-100)
was then added to the sample. The rabbit polyclonal anti-gC (R46)
and anti-gD (R7) antibodies were added to a final dilution of
{fraction (1/40)}. The mixture was incubated at room temperature
for 1 h with gentle rocking. Forty .mu.l slurry of Protein
A-agarose beads (Sigma Chemical Co., St. Louis, Mo.) was added. The
mixture was further incubated at room temperature for 1 h. The
Protein A beads were recovered by a 10 second centrifugation and
washed 4.times. with 1 ml of bead wash buffer (50 mM Tris, pH 7.5,
150 mM NaCl, 5 mM EDTA). The beads were then boiled with 50 .mu.l
of SDS-PAGE buffer and the proteins were analyzed on a 7.5%
SDS-PAGE gel (Laemmli, 1970) along with the high molecular weight
protein markers (Bio-Rad Laboratories Ltd., Mississauga, ON).
Following electrophoresis, the gel was fixed in 30% ethanol-10%
acetic acid for 1 h. The gel was then washed with distilled water
for 2.times.15 min and incubated with 1 M sodium salicyclic acid
for 30 min. The gel was then drier with a gel dryer and
autoradiographed on X-ray films (X-Omat, Kodak). The relative
molecular masses of the protein bands were estimated from the
Coomassie blue stained protein markers.
[0177] Identification of gC and gD as PVP binding targets. To
identify the HSV-1 glycoproteins that interact with the PVP
complex, the viral cultures were radiolabelled with
.sup.35S-methionine, lysed with detergents and the solubilized
proteins were subjected to affinity chromatography on a
PVP-Sepharose column. The bound proteins were eluted with PVP
complex and immuno-precipitated with anti-gC and and gD antibodies.
The immuno-precipitate was recovered by protein A-agarose beads and
analyzed by SDS-PAGE and autoradiography. As shown in FIG. 11A, two
broad protein bands of 110-140 kDa and 48-65 kDa were present in
the PVP complex eluate. Two of protein bands (110 kDa and 65 kDa)
were isolated by imuno-precipitation with anti-gC antibody and a 60
kDa protein band was immunoprecipitated by the anti-gD antibody.
These immuno-precipitated bands were absent from samples prepared
from mock-infected Vero cell lysates (FIG. 11B).
Example 19
The in Vivo Anti-HSV-1 Activities of PVP Complex
[0178] This example is to determine the anti-HSV-1 efficacy of PVP
complex creams in a guinea pig skin lesion model.
[0179] Material. Testing samples are PVP complex cream, topical use
preparation, in brown color, 50 g/vial (Batch no. 200205), provided
by the Department of Biology, the Chinese University of Hong Kong,
Hong Kong. Positive drug control agent is 3% Acyclovir cream, white
milky paste, 10 g/tube (Batch no. 991002, ZWYZZ (1996)-214602),
provided by Wenzhou Pharmaceuticals of Zhejiang, China. Virus is
Herpes simplex type 1 (HSV-1, Strain no. SM44), imported from The
American Type Culture Collection. The virus strain was proliferated
and preserved by the Department of Virological Diagnosis,
Institution of Virology, Chinese Preventative Medical Academy.
"Bunched needle" refers to seven individual needles (size No. 7)
bunched together as one bunch and seven bunches were put together
as one "Bunched needle". Other related apparatuses: All other
apparatuses and reagents were provided by the Department of
Virological Diagnosis, Chinese Preventative Medical Academy.
[0180] Animals: Guinea pigs, half male and half female, 200-250
grams (animal license no. YDHZ SCXK11-00-0006), supplied by the
Animal Center of the Chinese Medical Academy and Peking Union
Medical College.
[0181] Toxicity evaluation of the testing samples. The zero toxic
dose (TD.sub.0) of testing PVP complex cream was 15 g % per animal
for topical application (Data were provided by the Department of
Biology, the Chinese University of Hong Kong, Hong Kong).
[0182] HSV-1 titer determination. The dorsal hairs of guinea pigs
were removed with 8% barium sulfide (BaS) and the naked areas (40
cm.sup.2) were washed with 37.degree. C. warm water and then dried
with tissue paper. The naked dorsal skins were stabbed with a
"bunched needle", and the stabbed area was divided into 4 areas.
The areas were infected with 4 concentrations of virus suspension
(10.sup.-0, 10.sup.-1, 10.sup.-2 and 10.sup.-3 dilutions). Each
diluted virus suspension was applied to 5 guinea pigs. Each skin
area was infected with 30 .mu.l of the diluted virus suspension.
The animals were observed for 10 days for the development of
lesions. The median infectious dose (ID.sub.50) was then calculated
on the basis of the infected areas with symptoms versus the entire
infected dorsal skin area. The median infectious dose (ID.sub.50)
of HSV-1 (strain SM 44) to guinea pigs was determined to be
10.sup.-1.5.
[0183] HSV-1 cutaneous lesion in guinea pig. The animals were
grouped in 5. Animals within each group will be the same gender and
have similar body weight. The hair was removed from dorsal side of
the animal and the skin was abraded with needles as described
above. The entire abraded area was infected with 150 .mu.l of HSV-1
stock suspension.
[0184] Typical herpes lesions appeared on the infected area on the
4th day of infection. The extent of pathological changes was scored
as described below. PVP complex, acyclovir, or base creams were
then applied with cotton swabs on day 4 to the infected area. The
amount of cream given to each animal was 1.5 gram per dose twice
daily for a six-day duration.
[0185] Daily records of the pathological changes infected area were
made according to the following criteria:
[0186] When the skin lesions developed up to 1/4 of the total area,
the pathological changes were scored as 1.0-1.6;
[0187] When the skin lesions developed on {fraction (2/4)} of the
total area, the pathological changes were scored as 1.7-2.4;
[0188] When the skin developed on 3/4 of the total area, the
pathological changes were scored as 2.5-3.2;
[0189] When the skin lesions developed {fraction (4/4)} of the
total areas, the pathological changes were scored as 3.3-4.0;
[0190] When the skin herpes scars developed up to 1/4 of the total
areas, the pathological changes were scored as 0.9-1.0;
[0191] When the skin herpes scars were up to {fraction (2/4)} of
the total areas, the pathological changes were scored as
0.7-0.8;
[0192] When the skin herpes scars were up to 3/4 of the total
areas, the pathological changes were scored as 0.5-0.6;
[0193] When the skin herpes scars were up to {fraction (4/4)} of
the total areas, the pathological changes were scored as
0.2-0.4;
[0194] When the skin herpes scars were all detached and lesions
healed, the pathological changes were scored as 0.
[0195] Four days after infection, herpes lesions were observed on
dorsal skins of the animals. The extent of pathological changes
(EPC) was >3.8 out of 4.0 (Table 8). The animals were then given
treatment twice daily for six days. A greater reduction in EPC was
observed in animals that received PVP complex than the base cream
group. In the 15% PVP group, a significant reduction in EPC was
observed after 3 days of treatment as compared to the base cram. By
day 11, the EPC was very small in the 15% PVP group. A similar
pattern of EPC reduction was observed in the acyclovir control
group. The inhibition rates calculated from the day 11 data for PVP
cream treatment groups to pathological changes were 90%, 58%, 33%
and 6%, respectively, while that of the acyclovir was 96%. The
therapeutic effects of PVP complex and acyclovir was convincing
when the data was compared to the virus control group in which the
animals was infected but received no treatment. In this control
group, typical herpes lesions appeared on the dorsal skin areas on
the 4th to 6th days of viral infections with HSV-1. The extent of
pathological changes was observed at an average value of 3.94 with
blisters on most areas. In the 15% PVP group, the extent of lesions
began to decrease on day 6 and scabs were appeared on the 4th day
of drug application. All herpes lesions had formed scabs at the
sixth day of drug application. In the virus control group, some
animals showed the signs of paralysis in their lower limbs,
indicating that the virus had invaded into the animal lower limb
nerves with severe viral infections. The signs of paralysis were
not observed in the PVP and acyclovir groups.
[0196] PVP complex cream was found to have significant in vivo
anti-HSV-1 effects. Following a six-day treatment course, not only
the morbidity was decreased, the HSV-1 infection course was also
shortened. The potent therapeutic effects were comparable to
acyclovir at the concentration tested. These results strongly
suggest that PVP complex can be developed into an effective
anti-HSV drug.
8TABLE 8 Therapeutic effects of PVP complex creams on HSV-1 skin
lesions in guinea pigs Extent of Skin Pathological Changes (Mean
.+-. SD) and Inhibition rate Agent Skin Area Day 4* Day 5 Day 6 Day
7 Day 8 Testing Dose No. of (width .times. EPC IR EPC IR EPC IR EPC
IR EPC IR Group (g %) Animals length.) (cm.sup.2) (%) (cm.sup.2)
(%) (cm.sup.2) (%) (cm.sup.2) (%) (cm.sup.2) (%) PVP 15 15 6
.times. 7 3.85 .+-. 0.15 0 3.78 .+-. 0.14 5 3.39 .+-. 0.10 14 2.74
.+-. 0.16 25 2.10 .+-. 0.26 36 Cream 10 15 6 .times. 7 3.83 .+-.
0.16 0 3.83 .+-. 0.14 3 3.57 .+-. 0.19 9 3.05 .+-. 0.43 16 2.57
.+-. 0.28 22 5 15 6 .times. 7 3.84 .+-. 0.15 0 3.85 .+-. 0.11 3
3.75 .+-. 0.09 5 3.39 .+-. 0.17 7 3.13 .+-. 0.27 5 2.5 15 6 .times.
7 3.81 .+-. 0.18 0 3.78 .+-. 0.16 5 3.71 .+-. 0.18 6 3.41 .+-. 0.20
6 3.15 .+-. 0.16 4 Base 15 6 .times. 7 3.82 .+-. 0.02 3.82 .+-.
0.02 3 3.99 .+-. 0.01 0 3.99 .+-. 0.01 0.02 3.58 .+-. 0.04 3 cream
Acyclovir 3 15 6 .times. 7 3.87 .+-. 0.12 0 3.54 .+-. 0.22 11 2.77
.+-. 0.40 30 2.02 .+-. 0.30 44 1.21 .+-. 0.38 63 Virus 15 6 .times.
7 3.84 .+-. 0.14 3.96 .+-. 0.05 3.94 .+-. 0.07 3.63 .+-. 0.20 3.29
.+-. 0.26 Extent of Skin Pathological Changes (Mean .+-. SD) and
Inhibition rate Statistical Day 9 Day 10 Day 11 Treatment Testing
EPC IR EPC IR EPC IR T P Datum Group (cm.sup.2) (%) (cm.sup.2) (%)
(cm.sup.2) (%) value value Analysis PVP 1.32 .+-. 0.36 54 0.69 .+-.
0.24 71 0.20 .+-. 0.17 90 12.25 <0.01.sup.a Significant Cream
therapeutic effects 1.87 .+-. 0.55 35 1.28 .+-. 0.53 47 0.86 .+-.
0.36 58 5.74 <0.01 Significant therapeutic effects 2.71 .+-.
0.36 6 2.17 .+-. 0.50 10 1.37 .+-. 0.26 33 3.83 <0.01
Therapeutic effects 2.85 .+-. 0.16 1 2.55 .+-. 0.30 0 1.93 .+-.
0.34 6 0.64 >0.05 No therapeutic effects 3.16 .+-. 0.01 4 2.66
.+-. 0.03 5 2.06 .+-. 0.03 7 1.8 >0.05 No therapeutic effects
Acyclovir 0.82 .+-. 0.16 71 0.38 .+-. 0.18 84 0.09 .+-. 0.19 96
12.47 <0.01 Significant therapeutic effects Virus 2.87 .+-. 0.35
2.40 .+-. 0.34 2.06 .+-. 0.30 Control EPC: Extent of pathological
changes; IR: Inhibition rate = (Lesion area of virus control group
- Lesion area of test group) / Lesion area of virus control group
*The typical herpes lesions on dorsal skins of guinea pigs
developed to approximate 3.84 cm.sup.2 at the 4th day of infection
with HSV-1. Treatment was then given twice daily for six days.
Observations continued to the eleventh day after drug treatment was
suspended. T values of statistical treatments were calculated from
the 11th day data. .sup.ap value from Student's t test between the
test group and the virus control group.
Example 20
The in Vivo Anti-HSV-2 Activities of PVP Complex
[0197] This example is provided to determine the therapeutic
efficacy of PVP complex creams against herpes simplex virus 2
(HSV-2) infection in a mouse vaginal infection model.
[0198] Materials: Testing samples include 5%, 10% and 15% PVP
complex creams in brown color, 50 g/vial. (Batch no. 200205),
provided by the Department of Biology, the Chinese University of
Hong Kong, Hong Kong. Positive drug control agent is 3% Acyclovir
cream, white milky paste, 10 g/tube (Batch no. 991002, ZWYZZ
(1996)-214602), provided by Wenzhou Pharmaceuticals of Zhejiang,
China. Virus is Herpes simplex type II (HSV-2, Strain no. 333,
batch no. 200104010), provided by the Department of Virological
Diagnosis, Institution of Virology, Chinese Preventative Medical
Academy. Needles for vaginal tract infection (size no. of 12) were
prepared by the Department of Virological Diagnosis, Chinese
Preventative Medical Academy. African green monkey kidney cells
(Vero cells) were provided by the Department of Virological
Diagnosis, Chinese Preventative Medical Academy. Physiological
saline and cell culture medium was provided by the Department of
Virological Diagnosis, Chinese Preventative Medical Academy.
Inverted microscope was provided by the Department of Virological
Diagnosis, Chinese Preventative Medical Academy. Carbon dioxide
incubator was provided by the Department of Virological Diagnosis,
Chinese Preventative Medical Academy. And other related
apparatuses: provided by the Department of Virological Diagnosis,
Chinese Preventative Medical Academy.
[0199] Animals: BALB/c mice, female, body weight range of 18-20
grams. Animal license no. YDHZ 01-3003, supplied by the Animal
Center of the Chinese Medical Academy and Peking Union Medical
College.
[0200] Toxicity evaluation of the PVP. The zero toxic dose
(TD.sub.0) of testing PVP complex cream was 15 g % per mouse for
topical application (Data were provided by the Department of
Biology, the Chinese University of Hong Kong, Hong Kong).
[0201] Virus titer determination. HSV-2 stock was diluted from
10.sup.-0 to 10.sup.-3. The diluted virus suspension (0.03 ml) was
injected into the vagina by inserting the needle in a rotatory
manner with a stabbing motion along the vaginal wall. Each dilution
of virus was inoculated to 10 mice per group. The mice were kept
under observation for 12 days, by which time the animals developed
vaginitis or lethality occurred. The median lethal dose (LD.sub.50)
was then calculated. With a parallel group, vaginal samples were
taken at different time of viral infection. The virus titers
(median tissue culture-infective dose, TCID.sub.50) of the samples
collected at the respective infection stages were determined by
culturing on Vero cell.
[0202] In Vivo Anti-HSV-2 Activities of PVP Complex Creams:
[0203] 1. Target of the experiments: The mice could develop viral
vaginitis following infection with HSV-2. After treatment with the
PVP complex creams, the morbidities, mortalities, average surviving
days, protection rates, and life span extension rates were
calculated. Statistical analysis, t values and p values, was
calculated in comparison with the virus control group. Efficacy of
the testing samples will be evaluated on the basis of statistical
calculation. The median lethal dose (LD.sub.50) of HSV-2 stock was
determined to be 10.sup.-2.35. The virus titer (50% tissue
culture-infective dose, TCID.sub.50) of the vaginal tract samples
after viral infection was determined to be 10.sup.-2.35 by Vero
cell culture.
[0204] 2. Infection with virus: The animals were infected with a
virus dose of 10 LD.sub.50 in using the method as described above.
The BALB/c mice were divided into 3 test groups (5, 10, and 15% PVP
complex), one positive control (acyclovir), one negative control
group (base cream), and one no-treatment (virus control) group. The
number of animals per group was 10. Ten additional animals that
were not infected with HSV-2 and received no treatment served as
the uninfected control group.
[0205] Symptoms of viral vaginitis were observed on the third day
of infection. The symptoms were topical edema of vaginal tracts
with turbid secretions. Treatment began on day 3 post-infection.
This was achieved by applying the cream to the vaginal tract with
cotton swabs. The creams were given at a dose of 2 mg per mouse
twice daily for a six-day duration. Mortality and the number of
days for lethality to occur were recorded. The experiment was
repeated twice. The protection rates and life span extension rates
were calculated. The efficacy of testing agents were judged in
comparison with the positive control group on the basis of the
calculated t values and p values.
[0206] Viral vaginitis was observed on the third day following
HSV-2 infection. The survival rate and number of days for lethality
to occur were recorded. The latter was expressed as the average
life span in days.
9TABLE 9 Anti-HSV-2 efficacy of PVP complex creams in a mouse
genital infection model. Median Median No. of Average Effective
Life Span Effective Agent Dose animals No. of No. of Survival Life
Span Protection Dose Extension Dose (g %) (n) survival death
(%).sup.b (Day).sup.c Rate (%).sup.d (ED.sub.50) Rate (%).sup.c
(ED.sub.50) PVP Cream 15 30 22 8 73.3 11.2 .+-. 1.33 71.4 6.62 75
5.32 (p < 0.01).sup.f (p < 0.01) 10 30 19 11 63.3 10.7 .+-.
1.82 60.7 67.2 (p < 0.01) (p < 0.01) 5 30 14 16 46.7 9.5 .+-.
2.50 42.9 48.4 (p < 0.01) (p < 0.01) 3% 30 24 6 80 11.6 .+-.
0.96 78.6 81.2 Acyclovir (p < 0.01) (p < 0.01) Base cream 30
4 26 13.3 7.2 .+-. 2.07 7.1 12.5 (p > 0.05) (p > 0.05)
Uninfected 30 30 0 100 Control Virus 30 2 28 6.7 6.4 .+-. 1.73
Control.sup.a .sup.aAnimals infected with the virus but received no
treatment. .sup.bSurvival = (Number of survival / Number of
animals) .times. 100. .sup.cAverage life span of animals that died.
.sup.dProtection rate = (Survival of virus control group - Survival
of test group) / Survival of virus control group. .sup.eLife sapn
extension rate = (Average life span of test group - Average life
span of virus control group) / Average life span of virus control
group .sup.fp value from Student' t test between the test group and
the virus control group.
[0207] As shown in Table 9, the animals that received the PVP
complex cream showed a dose-dependent survival rate with the
highest survival at the 15% concentration. The survival rate for
the 15% PVP complex group is comparable to that of 3% acyclovir.
Animals received the base cream showed a very low survival rate.
The average life span of the animals that died from the infection
was highest for the 15% PVP complex and the acyclovir groups. The
protection rates of the three concentrations of PVP complex were
calculated to be about 71.4%, 60.7% and 42.9%, respectively. The
median effective dose (ED.sub.50) for protection was about 6.62%.
The life span extension rates were calculated to be about 75%,
67.2% and 48.4%, respectively. The median effective dose
(ED.sub.50) for life span extension was about 5.32%. The protection
rate of 3% acyclovir was 78.6%, and the life span extension rate
was 81.3%. The results showed that PVP complex has an excellent
therapeutic effects against HSV-2 infection in the mouse model.
[0208] 3. Recovery of HSV-2 from infected animals. The animals were
infected as above. Symptoms of viral vaginitis in the infected mice
were observed on the third day infection. Before treatment, vaginal
tract samples were collected with cotton swabs and transferred to
0.5 ml of physiological saline and stored at -25.degree. C. The
animals were treated with the creams as above for 6 days. One day
following the completion of the treatment, vaginal samples were
obtained and were kept at -25.degree. C. Samples were also obtained
from dead animals. In a parallel group, vaginal samples were taken
at different times of viral infections. The vaginal samples were
diluted by 1/5 in cell culture medium and used to infect Vero
cells.
[0209] The presence of HSV-2 on the vaginal samples was determined
by cytopathic effect (CPE) to Vero cells. The extent of CPE shown
in each sample will be observed and recorded. "+"=up to 25% cells
showing CPE; "++"=26.about.50% cells showing CPE;
"+++"=51.about.75% cells showing CPE; "++++"=76.about.100% cells
showing CPE. The experiment was repeated twice.
[0210] The effect of PVP complex treatment on the clearance of
HSV-2 was assessed from vaginal sampling before and after
treatment. The results are shown in Table 10.
10TABLE 10 Therapeutic effects of PVP complex treatment on the
reduction of HSV-2 in the genital tract. No. of No. of samples
Statistical vaginal containing HSV-2 Calculation samples Before
After Therapeutic (After Treatment) Agent Dose (g %) (1/mouse)
Treatment Treatment Rate (%).sup.a X.sup.2 p Value Data Analysis
PVP Cream 15 30 30 9 68 23.3 p < 0.01.sup.b Significant
antiviral effects 10 30 30 16 42 12.3 p < 0.01 Antiviral effects
5 30 30 21 25 5.3 p < 0.01 Antiviral effects Acyclovir 3 30 30 8
71 27.8 p < 0.01 Significant antiviral effects Base cream 30 0
27 4 0.22 p > 0.05 No antiviral effects Uninfected 30 0 0
Control Virus Control 30 30 28 .sup.aTherapeutic rate = (Percent of
HSV-2 positive samples in virus control group - Percent of HSV-2
positive samples in test group) / Percent of HSV-2 positive samples
in virus control group HSV-2 after treatment .sup.bp value from
Student's t test between the test group and the virus control
group.
[0211] The animals that received 15, 10 and 5% PVP complex showed
therapeutic rates of about 68, 42 and 25%, respectively. The
highest rate was comparable to that of animals received 3%
acyclovir. These results indicate that PVP complex treatment
reduced the HSV-2 load in the genital tract and correlate well with
the survival data reported in Table 9.
[0212] On the basis of experimental results, it was concluded that
5% and higher concentrations of PVP complex creams have in vivo
anti-HSV-2 therapeutic effects.
Example 21
In Vivo Toxicity Testing of Anti-Herpes Compound(s) from Prunella
vulgaris
[0213] The in vivo toxicity of invention compound can be tested
using means known in the art, for example, a two-step procedure on
albino mice as follows:
[0214] Step 1: Dose Ranging Determination.
[0215] To determine the dose which will produce a toxic effect in
mice, the anti-herpes extract PVP dissolved in 0.4 ml of distilled
water was administered orally, via a feeding tube, to BALB/c female
mice (8 month-old, 22 to 23 gram). A low dose (e.g., 25 mg/kg) of
the new compound is administered orally to one animal which is then
observed hourly for 24 hours and thereafter, every eight hours,
with continuous monitoring daily for 14 days. A second animal
receives double the dose of the first animal. The process is then
repeated for subsequent animals, each receiving a dose twice that
of the previous animal to a maximum of 2000 mg/kg (OCED guidelines
1995). A total of ten animals are used to establish dosing in this
step. The amount of PVP administered per animal was 25, 100, 200
400 and 800 mg/kg body weight.
[0216] At each of the doses, the animals survived and showed no
signs of in a moribund state. A moribund state is characterized by
symptoms such as shallow, labored or irregular respiration,
muscular weakness or tremors, absence of voluntary response to
external stimuli, inability to remain upright, cyanosis and coma.
Any one of these indicators will mark a moribund condition, and the
animal is euthanized immediately. However, if no moribund effects
or lethality were observed, the animals are killed and selected
organs (heart, lung, liver, spleen, kidney, brain, muscle, uterus)
sampled for histopathology at day 14. Since no moribund effects or
lethality was observed, the animals were euthanized and the organs
of each animal were examined. Histological examination of the
selected organs showed no inflammation or any other pathological
signs. These results indicate that PVP has no in vivo toxicity at a
concentration up to 800 mg/kg.
[0217] Step 2: Approximate Acute Toxicity (LD.sub.50)
Determination.
[0218] Using dose ranging determinations from Step 1, three
appropriate dose levels are established for LD.sub.50
determination. Ten animals are used for each dose level, and
monitored hourly for 24 hours and thereafter, every eight hours,
with continuous monitoring daily for 14 days. Animals exhibiting
moribund signs are killed immediately; all animals are killed at 14
days and their organs sampled for histology. Using the data from
these experiments, actual LD.sub.50 is estimated by extrapolation
and mathematical manipulations using known methods in the art (see,
for example DePass. in Toxicology Letters 49:159 (1989).
[0219] Compounds present in the untreated extract (see Example 3)
and in the purified fraction (Example 5) are active in suppressing
the cytopathogenic effects of HSV in vitro. Based on this latter
activity and certain of their biochemical characteristics
(particularly their anionic properties) these preparations of the
extract are capable of suppressing the cytopathogenicity of other
enveloped viruses. This extrapolation is supported by Baba et al
(see Antimicrobial Agents and Chemotherapy 32: 1742 (1988)).
[0220] In conclusion, it has been shown that the invention
compositions, obtained, for example, from the spikes of Prunella
vulgaris, are effective agents for the treatment of the
cytopathogenic effects of an enveloped virus in mammals. These
results provide insight into the pathway of action of the purified
extract fraction within enveloped viruses.
[0221] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
* * * * *